TR201815301T4 - Antimicrobial peptide and its uses. - Google Patents

Abstract

The invention relates to antimicrobial peptides, pharmaceutical compositions comprising the peptides and their use in the treatment or prevention of microbial, bacterial, fungal, viral and parasitic infection.

Description

DESCRIPTION ANTIMICROBIAL PEPTIDE AND ITS USES The invention relates to the field of biochemistry and medicine. More specifically, the invention with the field of antimicrobial peptides and bacterial, viral, fungal and parasitic related to the inactivation of infections.

Antimicrobial Peptides (AMPs) are the defense mechanisms of organisms throughout nature. system and offers protection from invading pathogens. Gram-positive` and potent against Gram-negative bacteria, fungi, parasites and viruses show antimicrobial activity. Majority of microbial cells What are the AMPs that work by disrupting the structure or function of their membranes? as small (usually about 15-40 amino acids) they do not target molecular structures. Therefore, conventional antibiotics on the contrary, they are effective regardless of the metabolic activity of the bacteria. Let them defend and Human AMPs such as cathelicidin (LL-37) are present in leukocytes and They are secreted by various epithelium on the surfaces. antimicrobial activities In addition, AMPs are involved in inflammation, immune activation and wound healing. are important effector molecules in healing. AMPSIer array and secondary structure They are quite diverse in terms of aspects, but they share some common features. Mostly They are cationic, amphipathic, and their germicidal effects, bacterial membrane they show by breaking their integrity. AMPalerin anionic membrane of target microbes surfaces, leading to membrane permeability, cell lysis and death. opens. In general, the cytoplasmic membrane is the main target of many AMPs. peptide accumulation in the membrane leads to increased permeability and barrier causes loss of function, which is accompanied by leakage of cytoplasmic components. and results in cell death.

Conventional antibiotics treat bacteria as an epitope on the cell wall. to targets or targets in bacterial protein and DNA or RNA synthesis it kills by binding. Pathogenic bacteria by modifying antibiotic targets 02116-P-0001 develop resistance faster so antibiotics can no longer bind to these targets.

A major advantage of AMPs over conventional antibiotics is that resistance is rapidly is not development. One reason for this is the use of single-definition antibiotics such as conventional antibiotics. does not target molecular structures (epitopes), but kills microorganisms function on the cell membrane. AMPs specifically, biofilm-associated infections (BAI), which are mostly bacteria but surface-attached cellular aggregates of microorganisms, including fungi It is useful in neutralizing infections. Biofilms, conventional contributes significantly to bacterial resistance to antibiotics. biofilms cystic fibrosis, colonization of permanent medical devices and dental plaque in humans It is associated with various pathological conditions such as formation and wounds. associated with biofilm inactivation of the infection with conventional antibiotics also stimulating the release of proinflammatory microbial compounds, biofilm inactivation in blood as a result of insufficient penetration and necessary systemic administration or is insufficient for a number of reasons, including deterioration. of AMPs rapid onset of killing other advantages over conventional bacteria, in a way that reduces existing concern about residual antibiotics in the environment that they are biodegradable and have concomitant anti-inflammatory activity fact is included.

Within the potential pool of hundreds of natural and synthetic peptides, relatively very few have entered clinical trials. Examples are magainin peptide, omiganan, OP- 145, novexatin, and lyticar. Only two AMPs, daptomycin and DPK-060 Clinical trials up to stage 2 for OP-145, also known as has been carried out. OP-145, endogenous human cathelicidin antimicrobial peptide LL- It is a 24 amino acid peptide derived from 37°. OP-145, chronic inflammation of the middle ear As an endotoxin-neutralizing antimicrobial peptide for topical treatment developed. In addition to being an anti-microbial peptide, OP-145 contains LPS. neutralizes. 02116-P-0001 The currently known AMPs, including the OP-145, still have a few flaws. has. For example, P. aeruginosa, E. faecali's, Proteus mirabili's, Streptococcus pyogenes and various bacteria such as S. aureus, such as cathelicidin LL-37 secretes proteases that dissolve many antimicrobial peptides. Thus, the protease resistant antimicrobial peptides are advantageous from a therapeutic point of view. Moreover, In addition to the potential lytic effect, AMP can be found in bacterial but also mammalian Due to its other properties against membranes, it is important to design new peptides. One of the challenges is bacterial or bacterial infection compared to the cellular membranes of the infected patient. It has high specificity against microorganisms such as fungal cells, that is, a high therapeutic index (minimal hemolytic concentration/minimal antimicrobial activity; It is based on the development of AMPs with MHC/MEC). OP-145 included Another important disadvantage of known AMPs, especially is strongly influenced by the presence of concentrations. For example, OP- concentration) LC99.9 about 200 µM°. This is a particular disadvantage for systemically administered AMPi.

Due to non-specific binding of AMP71s to plasma components, enzymatic by plasma components such as degradation or peptide absence. Significantly lower in the presence of plasma-like peptide inactivation Several mechanisms may be responsible for its activity. Resistant to plasma components AMPs not only because they are potential systemic therapeutic agents, but treatment of infected wounds and medical implant-related infection and inflammation is also important for Especially in deep tissue infections, proteolytic resistant highly proteolytic, which can lead to an inactivation of non-AMP°s. activity may be present. Excessive amounts of wound fluid from chronic wounds protease and implanted materials are rapidly released from host fluids. coated with plasma components. Both chronic wounds and medical implants are frequently associated with microbial biofilms. In particular, biofilm-associated infections Antibiotics are often given systemically in the treatment of and is prone to enzymatic dissolution in surrounding tissues. Thus, the plasma 02116-P-0001 Due to the sensitivity to its components, many AMPs, including the OP-l45 its applicability will be limited to, for example, topical applications. Because, especially with potential systemic therapeutic agents and/or for example chronic wounds and Effective therapeutic agent against biofilm infections associated with medical implants Additionally, there is an obvious need for alternative AMPs resistant to plasma components. It is an object of the present invention to overcome the shortcomings of conventional antibiotics. known for their resistance to plasma components. A new potent potentiator with enhanced properties over antimicrobial peptides. to provide antimicrobial peptides. Another purpose of the invention is that people like against non-allergenic, pathogenic microorganisms when administered to mammals pathogenicity in biofilm-associated infections with high specificity antimicrobial with high antimicrobial activity against microorganisms to provide peptides. Peptides and polypeptides of the invention are found in both biofilms. to both microorganisms and unorganized microorganisms in biosciences Applies potent, broad-spectrum antimicrobial activity against possesses antimicrobial activities and can be used as therapeutic, prophylactic or diagnostic agents. can be used as Peptides and polypeptides of the invention have a limited duration of action and designed to overcome limited applicability issues because their antimicrobial activities in blood, plasma and serum and their they preserve in the presence of their components.

The present inventors refer to the series LKKLYKRLVKILKRWWRYLKRPVR. P139-P163 peptides based on Gram-positive bacteria (e.g. Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative bacteria (e.g. Pseudomonas aeruginosa) as well as fungi (Candida albi'cans and Aspergillus niger), see Tables 2-4 and Figure 1 and 2. Peptides thus offer broad spectrum antimicrobial activity. All peptides with low IC99.9 values similar to those of OP-145° in PBS It is very strong as has been proven. Importantly, all peptides are plasma 02116-P-0001 It was found to be more effective than OP-145 in its presence. Most of the peptides are plasma significantly with up to 16-fold higher activity than OP-145 in its presence. was stronger (Tables 2-4). As set out in Table 2, OP- in the presence of plasma. P140, P141, artis) and has P149 and P155. It has been shown to be effective against (see Tables 2-4). In addition, P145, P148 and P159 can prevent S. aureus biofilm formation (see Figure 3). Moreover, P and as they neutralize lipopolysaccharides (LPS), thereby increasing the proinflammatory response. It has demonstrable immunomodulatory activity as they reduce it (Table 12).

In addition, shorter P148 variants with at least 16 amino acids can be found in PBS. It has also been found to have an antimicrobial activity similar to that of P1487. (see Table 5). Importantly, antimicrobial activity in the presence of plasma it is also retained in these shorter variants. 14 amino of P1485 reduced antimicrobial compared to a shorter variant P148* with acid activity, but still 5-fold greater than OP-145 in the presence of plasma is strong.

Thus, a polypeptide according to the present invention can exist both in the presence of plasma and It has high antimicrobial activity against micro-organisms in the absence of and optimal anti-inflammatory activity as evidenced by LPS and LTA neutralizing activity. found in biofilms with inflammatory (microbial compound-neutralizing) activity or not found. 02116-P-0001 Effect of antimicrobial peptides of the invention on biofilm infections multiplex: it will inhibit biofilm formation and disperse existing biofilms, bacteria, fungi, or other microbes in and around the release area and lipoteichoic acid (LTA), peptidoglycan (PG) and lipopolysaccharides (LPS) by neutralizing pro-inflammatory microbial endotoxins and phagocytic and activating macrophages to potentiate their germicidal activity. will plan their immune response. This immune control, implants to prevent surrounding tissue from becoming a new niche for pathogens is necessary.

Thus, the present invention is a LKKLYKRLVKILKRWWRYLKRPVR amino containing an acid sequence or a variant of said amino acid sequence. provides isolated or recombinant polypeptide antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic It has the same activity against at least one microbial species in the presence of 50% plasma. OP-l 45 (acetyl- a high in vitro antimicrobial, antibacterial, antiviral, antifungal and/or has antiparasitic activity, said variant sequence to at least 14 amino acids and optionally has: - 1 to 8” of the following amino acid substitutions: 0 One or more selected from group L, V, F, A, 1, W, Y or Q of the amino acid with another amino acid selected from the group in question. substitution; substitution of O R and/or Quinine with A or a positively charged amino acid; o one or more of an amino acid with a corresponding D-amino acid substitution; combining an amino acid with a corresponding non-natural amino acid 02116-P-0001 or more substitutes and the corresponding non-natural the amino acid is a derivative of the reference natural amino acid and/or . a residue of at least 14 consecutive amino acids from said amino acid sequence. is the inverse sequence, wherein said variant sequence is optionally one or more of the amino acid substitutions as defined above at least one KRLVKILKRWWRYL amino acid sequence has.

to amino acid sequences or variants thereof as defined herein. amino acids are represented by single-letter symbols. These one-letter symbols and three- letter symbols are well known to the skilled artisan and the following has the meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, l (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, Y (Tyr) is is tyrosine. As used herein, "a positively charged amino acid" pI-Ista, i.e. an amino acid with a positive charge at a pH of 7.3-7.47 is doing.

A polypeptide of the invention has antimicrobial activity, preferably antibacterial, antiviral and/or antifungal activity, more preferably antibacterial and/or It has antifungal activity. Also, a polypeptide of the invention is preferably both It has both antimicrobial and anti-inflammatory activity. used here As such, the term "antimicrobial activity" of a polypeptide means at least one microbe, for example, the growth of a bacterium, a virus and/or a fungus, or refers to the inactivation of proliferation and prevention, reduction or inhibition of proliferation as well as It also includes killing the microbe. A microbe, a microscopic one, that is, the majority! It is an organism that is too small to be seen with the naked human eye.

Microbes are very diverse bacteria, viruses, fungi, archaea, protozoans 02116-P-0001 and microscopic algae. Similarly, as used here inactivation of the growth or proliferation of a fungus and a parasite and their growth or proliferation prevention, reduction or inhibition, as well as their includes murder. Antimicrobial activity eg inhibitory concentration It is expressed as (1C) or lethal concentration (LC). used here as ICx or LCx, the lowest that kills at least x7% of germs after 2 hours stands for peptide concentration. For example, IC99.9 and LC99.9 microbes It refers to the lowest peptide concentration that kills 99.9% of the 2%.

Antimicrobial, antibacterial, antiviral, antifungal and antiparasitic activity, can be measured by methods known in the art.

One of these methods is detailed in the Examples of this application. and an in vitro assay for determination of antimicrobial activity. This In the method, microbes, for example bacteria or fungi, for example for 1-2 hours, It is incubated with different concentrations of a polypeptide according to the invention, then The microbe-polypeptide mixture was likewise incubated with the further processed polypeptide. surviving and/or killed compared to a sample of untreated microbes in a suitable culture medium for the determination of the number of microbes or is incubated on.

Virus plate for determination of antiviral activity of a polypeptide of the invention. appointments can be used. In short, a virus inoculum is a permissive cell single exposed to the polypeptide prior to infection of the layer. a standard After the interval, the Virus titer in the cellular extracts was higher than that of fresh cell monolayers. by infecting it and measuring its effects on the cell monolayer. determined using more than one dilution of the extracts. 02116-P-0001 For the determination of antiparasitic activity, a polypeptide of the invention and a parasite incubated for a standard time interval. Then the parasites its metabolic activity can be directly analyzed, for example, by an MTT assay or parasites are transferred to mammalian cells and after incubation parasite growth is determined by microscopy.

"anti-inflammatory activity" of a polypeptide as used herein, with germs, such as bacteria, viruses, fungi and/or parasites preventing, reducing, or reducing an inflammatory response in an infected subject means inhibition. Anti-inflammatory of the polypeptides of the invention activity, lipoteichoic acid (LTA), peptidoglycan (PG) and/or lipopolysaccharides By inhibiting salinacin of pro-inflammatory microbial compounds such as (LPS), It is obtained by reduction or inhibition. Anti-inflammatory activity can be measured by methods known in the art. Examples of these methods are An LPS neutralization assay and an LTA as described in the examples neutralization determination. In this method, a polypeptide of the invention is LPS or LTA. with a fixed concentration, eg 500 ng/ml LPS or 2 mg/ml LTA mixed and incubated for 30 minutes. Then these mixes diluted fresh human whole blood and 20 hours later level of cytokines (eg 1L-8 for LTA and 1L-12p40 for LPS) by ELISA The polypeptides of the invention are bound to plasma, also referred to as blood plasma, preferably resistant to human plasma. “plasma” or “human” as used herein plasma” has the common meaning used in the art. Blood, red and white blood cells and platelets are removed, proteins, hormones and other organic refers to the liquid part containing compounds and inorganic compounds such as electrolytes. is doing. “Plasma resistance” as used herein means 50% plasma, preferably against at least one microbial species in the presence of human plasma, under the same conditions. an in vitro at least 1.3-fold higher activity than OP-145 when determined antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic 02116-P-0001 defined as having activity. Preferably against at least 2 bicrobial species said activity is at least 1.5-fold higher than that of OP-145, more preferably at least 2-fold, more preferably at least 4-fold, more preferably at least 5-fold higher. Particularly preferred polypeptides are at least one against the microbial species, the antimicrobial of OP-1453 in the presence of 50% plasia It has an antimicrobial activity that is at least 16-fold higher than its activity. 145 Plasma in liquid such as PBS, preferably with the addition of human plasma, %SOalic means it is measured when incubated with microbes at a final concentration is coming. The expression "same reaction conditions" means that a polypeptide of the invention antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic The conditions under which its activity was measured, OP-145°in antimicrobial, antibacterial, the same conditions under which its antiviral, antifungal and/or antiparasitic activity was measured. means it is. Such conditions, not to be limited by them The buffer in which the polypeptide and microbes are incubated, provided that the microbial strain identity, concentration of microbes, incubation time and temperature, and used plasma source is included.

The OP-l45 has the sequence JIGKEFKRIVERIKRFLRELVRPLRB where J is acetyl and B is amide. A person skilled in the art will know that OP-1453in antimicrobial solid-phase synthesis commonly used with that of other peptides methods or recombinant methods described herein in more detail below. can synthesize OP-145 for comparison using techniques. Moreover, acetylation and amidation are common techniques used in the art, skilled in the art One therefore involves the acetylation of the N-terminus of the peptide chain of OP-145° and the C- As detailed herein, in vitro antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic activity preferably IC99.9 or IC50 is expressed as. “IC99.9” and “IC50” as used herein refer to a specific 02116-P-0001 period, e.g. at least 99.9% of the microbes, respectively, within 2 hours or Peptides of the invention are the same against at least one microbial species in the presence of 50% plasma. up to 75% of 99.9” of OP-145° when determined under It has an in vitro IC of 99.9°. According to the invention, in 50% plasma presence of a polypeptide, at least when determined in vitro IC99.9° against a microbial species under the same conditions at most 50%. more preferably at most 40%, more preferably at most 30%, more preferably at most 20%, most preferably at most 103%. Especially preferred polypeptides against at least one microbial species in 50% plasma presence, same conditions has.

Polypeptides of the invention preferably have at least one microbial species in the presence of 50% plasma It has an in vitro IC99.9 of up to 150 µM at 37°C after 2 hours versus 2 hours. promise subject IC99.9 preferably antimicrobial as described in the Examples herein. determined by a method for determining its activity. associated with biofilm for the determination of the antimicrobial activity of microbes that are not the described method was carried out in PBS at a final concentration of 50%. 50 microL of a polypeptide and bacterial of a solution of 20 microLysine of the suspension 5x106 per mL in one well. It involves mixing CFU with PBS. This mixture was heated at 37°C for 2 hours. After incubation under shaking conditions, determination of bacterial counts a sample is used. where 99.9% of bacteria are killed The low peptide concentration is called the inhibitory concentration (IC)99.9.

Bacterial counts can be determined by manual CFU counting. Antibacterial For example, lx10OCFU/ml bacteria are used to determine the activity, antifungal For example, 1X105 cells/ml are used to determine the activity.

For the determination of anti-biofilm activity, the subject matter described in the Examples The method shows that the polypeptide at 37°C at 1x108 CPU/ml S. aureus for 24 hours. 02116-P-0001 with JAR060131, with 20% plasma by overnight incubation at 4°C with plasma in biofilm-adjusted BM23 in coated 96-well polypropylene plates Determination of 1C505 after incubation of planktonic bacteria with PBS Four washes and staining of biofilms with crystal violet.

After solubilization with ethanol, the optical density at 590 nm is a fraction of the biofilm mass. determined as a measurement.

Polypeptides of the invention preferably up to 100 µM in the presence of 50% plasma, more preferably at most 80 µM°, more preferably at most 51.2 µMs, more preferably at most it has an in vitro IC of 99.9 at very 30 µM. Preferred polypeptides of the invention has an IC99.93a at most 25.6 µM as defined herein. Especially preferred polypeptides against at least one microbial species in the presence of 50% plasma it has an in vitro IC of 99.9° at up to 12.8 µM.

Said at least one microbial species is, for example, S. aureus, S. epidermidis, P.

a bacterial species such as Aeruginosa, a fungal species such as C. albi'cans and A. niger, A parasitic strain such as Plasmodiumfalci'parum and Toxoplasma gondii' or hepatitis A virus is a type of virus such as hepatitis C virus, Influenza A virus etc. Preferably, a polypeptide of the invention in the presence of 50% plasma at least one bacterial or fungal species, preferably S. aureus, S. epidermi'di's, P. aerugin'nosa, C. albi'cans and/or A. niger at most one in vitro IC of 99.9 at 105 µMs, most preferably a polypeptide of the invention in the presence of 50% plasma at least S. aureus, at least preferably Cainpoccia et al. (Int J Artif Organs. 2008 Sep;31(9):84l-7)* It has an in vitro IC of 99.9 at 105 µMs compared to S. aureussa. find it most preferred polypeptides against S. aureus as defined herein It has an IC of .6 µM, more preferably up to 12.8 µM.

A preferred variant sequence has at least one substitution of an amino acid with A.

Any of these amino acids, for example, at any of positions 1 to 24 It could be an amino acid. As set forth in Tables 6-87, an amino acid polypeptides that are substituted by alanine have at least one of their antimicrobial activities. 02116-P-0001 activity of some alanine-substituted polypeptides Also herein, a LKKLYKRLVKILKRWWRYLKRPVR amino acid sequence or an isolated or recombinant containing a variant of said amino acid sequence polypeptide is disclosed, said polypeptide is antimicrobial, antibacterial, It has antiviral, antifungal and/or antiparasitic activity and the same conditions At least 1.3-fold higher than the activity of OP-145 when determined under in vitro antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic has activity, said variant has at least 14 amino acids and is optionally has the following: 0 one or more of the following amino acid substitutions: o K° with a positively charged amino acid, preferably R, homolysine, homoarginine, omitin, diaminobutyric acid and diaminopropionic acid substitution; o R° with a positively charged amino acid, preferably K, homolysine, homoarinin, ornithine, diaminobutyric acid and diamino roionic acid substitution; o L replaced by V, 1 or W; o substitution of Y with W or Q; o substitution of V with F or A; o substitution of I" by L; o Substitution of W with F, Y, L or 1 - one or more of an amino acid with a corresponding D-amino acid substitution; . one of an amino acid with a corresponding non-natural amino acid, or more substitutions and/or 02116-P-0001 - a residue from said amino acid sequence of at least 14 consecutive amino acids eVirtik series.

Here also, a LKKLYKRLVKILKRWWRYLKRPVR amino acid sequence or an isolate containing a variant of said amino acid sequence, or recombinant polypeptide is disclosed, said polypeptide is antimicrobial, antibacterial, antiviral, antifungal and/or It has antiparasitic activity and when determined under the same conditions OP-145°in an in vitro antimicrobial at least 1.3-fold higher than its activity, has antibacterial, antiviral, antifungal and/or antiparasitic activity, said variant has at least 14 amino acids and is optionally has the following: 0 one or more of the following amino acid substitutions: o K° with A or a positively charged amino acid, preferably A, R, homolysine, homoarginine, ornithine, diaminobutyric acid and substitution with diaminopropionic acid; o R° with A or a positively charged amino acid, preferably A, K, honiolysin, homoarginine, omitin, diaminobutyric acid and substitution with diaminopropionic acid; o substitution of L with A, V, 1 or W; o substitution of Y with A, W or Q; o substitution of V with F or A; o substitution of I" by A or L; o Substitution of W with A, F, Y, L or 1 - one or more of an amino acid with a corresponding D-amino acid substitution; . one of an amino acid with a corresponding non-natural amino acid, or more substitutions and/or 02116-P-0001 - a residue from said amino acid sequence of at least 14 consecutive amino acids eVirtik series.

Such variant sequence also preferably has at most one substitution of an amino acid with A. has. It can be made from any amino acids, so 1 to 24 amino acids an amino acid at any of its positions may be substituted by A.

LKKLYKRLVKILKRWWRYLKRPVR amino acid as used herein A variant of the sequence has a length of at least 14 amino acids and preferably the following has one or more of the amino acid substitutions: 0 K with a positively charged amino acid, preferably R, homolysine, homoarginine, with ornithine, diaminobutyric acid and diaminopropionic acid, more preferably with R substitution; . R is with a positively charged amino acid, preferably K, homolysine, homoarginine, with ornithine, diaminobutyric acid and diaminopropionic acid, more preferably with K substitution; i substitution of L° with V, I or W; o substitution of Y with W or Q; - substitution of V7 by F or A; - substitution of I with L; - Substitution of Win with F, Y, L or 1 . substitution of one or more amino acids with the corresponding D-amino acid . one or more of the acids with a corresponding non-natural amino acid substitution. Preferably, up to said variant sequence 14 substituent, more preferably up to 10" to said substitution, eg 9°, or up to 1° of said substitution.

Preferably, said variant sequence optionally contains the following amino acid has one or more of its substitutions: 02116-P-0001 - substitution of K at 2 amino acid positions by R . Substitution of K by R at 3 amino acid positions . Substitution of U by V at the 4 amino acid position . Substitution of Y with W at the 5 amino acid positions . Substitution of L° with V at 8 amino acid positions . Substitution of V by F or A at the 9 amino acid positions i Substitution of Cl at the 10 amino acid position by R . substitution of 1 by L at 11 amino acid positions o Substitution of Ls at 12 amino acid positions by I or W . Substitution of W by F, Y, L or 1 at the 15 amino acid positions - substitution of W by F, Y, L or 1 at 16 amino acid positions . Substitution of Y by Q at the 18 amino acid position . Substitution of K by R at the 20 amino acid position o Substitution of R at the 21 amino acid position with K . substitution of one or more amino acids with a corresponding D-amino acid Here, the numbering of amino acid positions is as follows: Said variant is also preferably optionally combined with an amino acid A. has one or more, more preferably at most, one substituent. This, can be made with any amino acid, so amino acids 1 to 24 an amino acid at any of the extreme positions may be substituted by A.

Preferably, a variant sequence as defined herein is for the amino acid in question. up to 15" of substitutions, more preferably of said amino acid substitutions It has °. Also, a variant sequence as defined herein is preferably the most at least one KRLVKILKRWWRYL amino acid sequence, ie between 6 and 19 amino acids optionally one or more of said amino acid substitutions. has more. So here is a LKKLYKRLVKILKRWWRYLKRPVR 02116-P-0001 containing an amino acid sequence or a variant of said amino acid sequence An isolated or recombinant polypeptide is provided, said polypeptide is antimicrobial, antibacterial, antiviral, antifungal and/or It has antiparasitic activity and when determined under the same conditions, OP-145 an in vitro antimicrobial at least 1.3-fold higher than its activity, has antibacterial, antiviral, antifungal and/or antiparasitic activity, said variant sequence, optionally having at least one KRLVKILKRWWRYL has the amino acid sequence: 1 to 8 of the following amino acid substitutions: 0 One or more selected from group L, V, F, A, 1, W, Y or Q of the amino acid with another amino acid selected from the group in question. substitution; substitution of 0 R and/or K° with A or a positively charged amino acid; i one or more of an amino acid with a corresponding D-amino acid substitution; o one of an amino acid with a corresponding non-natural amino acid, or more substitutions; - a residue from said amino acid sequence of at least 14 consecutive amino acids inverted series.

A variant sequence having at least one of said amino acid substitutions is also preferable.

A preferred polypeptide according to the invention is P139, P140, as shown in Table 17. contains an amino acid sequence because these peptides are in PBS compared to the OP-145 peptide. It has high antimicrobial activity and increases in the presence of plasma. 02116-P-0001 has antimicrobial activity. Preferably, said polypeptide is in Table 1 contains an amino acid sequence of peptides because these peptides are OP- It has at least 4-fold increased activity compared to 145. More preferably, the sequence because these peptides are at least 8-fold in the presence of plasma compared to OP-1455e. has increased activity.

A particularly preferred polypeptide of the invention is the amino acid sequence of the P145 peptide. or a variant of said amino acid sequence It contains the LKRLYKRLAKLIKRLYRYLKKPVR ainino acid sequence. The variant sequence in question has at least 14 amino acids and optionally an amino acid acid with a corresponding D-amino acid and/or a corresponding non-natural has one or more substitutions by amino acid and/or optionally at least from said amino acid sequence of 14 consecutive amino acids to a reverse-inversion sequence more preferably said polypeptide At least 14-19 aininos of the amino acid sequence LKRLYKRLAKLIKRLYRYLKKPVR acids, most preferably said polypeptide It contains the amino acid sequence LKLRYKRLAKLIKRLYRYLKKPVR. This type of polypeptide It is particularly preferred because the P145 peptide is both in the presence of serum and potent, broad-spectrum antimicrobial activity and anti- has inflammatory properties.

Another particularly preferred polypeptide of the invention is the amino acid peptide P148. amino acid sequence LKRVWKRVFKLLKRYWRQLKKPVR, or comprising a variant of said amino acid sequence, said variant the sequence has at least 14 amino acids and optionally the reciprocal of an amino acid with a corresponding D-amino acid and/or a corresponding non-natural amino acid or more substitutions and/or optionally at least 14 consecutive amino acids 02116-P-0001 the acid has a reverse-inverting sequence from said amino acid sequence, more preferably said polypeptide LKRVWKRVFKLLKRYWRQLKKPVR amino contains at least 14-19 amino acid sequences of the acid sequence, most preferably said polypeptide LKRVWKRVFKLLKRYWRQLKKPVR, WKRVFKLLKRYWRQLK contains the amino acid sequence KRVFKLLKRYWRQL. is the amino acid sequence. This type of polypeptide is particularly preferred because the Pl48 peptide strong, broad-spectrum in both the presence and absence of serum has antimicrobial activity and anti-inflammatory properties, and P325, P326, has kept.

Another particularly preferred polypeptide of the invention is the amino acid peptide P159. amino acid sequence LKLRYKRVFRLLKRYYRQLRRPVR, or comprising a variant of said amino acid sequence, said variant the sequence has at least 14 amino acids and optionally the reciprocal of an amino acid with a corresponding D-amino acid and/or a corresponding non-natural amino acid or more substitutions and/or optionally at least 14 consecutive amino acids the acid has a reverse-inverting sequence from said amino acid sequence, more preferably amino acid of said polypeptide LKLRYKRVFRLLKRYYRQLRRPVR contains at least 14-19 amino acid sequences of the sequence, most preferably said polypeptide It is particularly preferred because the P159 peptide can be detected both in the presence and absence of serum. potent, broad-spectrum antimicrobial activity and anti-inflammatory has features.

Another preferred polypeptide according to the invention is shown in Tables 6, 7 and Side. 02116-P-0001 sequence because these peptides have high antimicrobial activity in PBS and It has increased antimicrobial activity compared to OP-145° in the presence of plasma. This Polypeptides with a type of amino acid sequence in which one amino acid is substituted by A. P145 are variants of the P148 or P159 polypeptides. find especially preferred a polypeptide of P148, in which R at position 7 is substituted by A. It has the amino acid sequence of the P276 peptide, which is a variant of it.

A preferred polypeptide according to the invention is from Tables 1, 2, 5, 6, 7, 8, 9 and/or 10°. has an amino acid sequence of a selected polypeptide. A polypeptide according to the invention has a lethal concentration (LC) of 99.9” LKKLYKRLVKILKRWWRYLKRPVR amino acid sequence or a contains the variant. In one embodiment, 50% plasma, selected from tables 2 or 5-10” a polypeptide having an LC of 99.9" up to 51.2 µM, more preferably up to 51.2 µM is provided.

Substitutions of one amino acid with another amino acid as described above. alternatively or in addition, as defined herein A variant of the amino acid sequence LKKLYKRLVKILKRWWRYLKRPVR is an L- with the corresponding D-amino acid of the amino acid or the amino acid shown above the amino acid in question after one or more of its substitutions one in the D-amino acyl corresponding to an L-amino acid present in the sequence or may contain more substitutions. Here is a capital single-letter such as A for alanine. The amino acids indicated by the symbol are commonly found in naturally occurring proteins. are L-amino acids. As used herein, the “corresponding D-amino acid” is defined as the D-amino acid equivalent of an L-amino acid. For example, 02116-P-0001 the corresponding D-amino acid of alanine (A) is D-alanine (a), the corresponding D-alanine (R) the corresponding D-amino acid is D-arginine (r), the corresponding D-aniino acid of asparagine (N) D-asparagine (n) Etc. All L- of a variant sequence as defined herein amino acids may be substituted by the corresponding D-amino acids. Therefore, wherein a LKKLYKRLVKILKRWWRYLKRPVR amino acid or Disclosed is a polypeptide comprising a variant of the amino acid sequence of interest, the said polypeptide is antimicrobial, antibacterial, antiviral, antifungal, has antiparasitic and/or anti-inflammatory activity, said variant sequence has at least 14 amino acids and is optionally has the following: . one or more of the following amino acid substitutions: 0 One or more selected from group L, V, F, A, 1, W, Y or Q of the amino acid with another amino acid selected from the group in question. or a corresponding amino acid of L, V, F, A, I, W, Y or Q substitution by D-amino acid; o R7 with a positively charged amino acid, preferably K, homolysine, homoarginine, omitin, diaminobutyric acid and diaminopropionic acid or substitution of K or R° with the corresponding D-amino acid; o K° with a positively charged amino acid, preferably R, homolysine, homoarginine, ornithine, diaminobutyric acid and diaminopropionic acid or substitution of R or K with the corresponding D-amino acid; o one or more of an amino acid with a corresponding D-amino acid substitution. Such variant sequence may also be accompanied by A or a corresponding D-amino one or more amino acids selected from acid-substituted K and R, preferably it has substitution of at most one amino acid. Preferably, that variant sequence optionally to one of the following amino acid substitutions or has more: 02116-P-0001 o substitution of K" for R or R" with the corresponding D-amino acid; o substitution of R by K or K9 by the corresponding D-amino acid; o the corresponding amino acid V, 1 or W or V, 1 or W of L substitution with a D-amino acid; o A corresponding W or Q or W or Q amino acid substitution by D-amino acid; o Vinin F or A or a corresponding D- of amino acid F or A substitution by amino acid; o With the corresponding D-amino acid of the L or L amino acid of isnin substitution; o The corresponding amino acid F, Y, L or I or L, F, Y, L or 1 of W° substitution with a corresponding D-amino acid; one or more of an amino acid with a corresponding D-amino acid substitution.

More preferably, said variant sequence optionally contains the following amino acid has one or more of its substitutions In the 2-amino acid subject of K7, R or the corresponding D-amino of R acid substitution; At the 3 amino acid position of quinine, R or the corresponding D-amino acid substitution; At the 4 amino acid positions of L, the corresponding D-amino of V or V* acid substitution; W at the 5 amino acid positions of Y or the corresponding D-amino of W° acid substitution; V or the corresponding D-amino at the 8 amino acid positions of L acid substitution; at the 9 amino acid positions of V, corresponding to F or A or F or A substitution with a D-amino acid; 02116-P-0001 - R at the 10 amino acid position of K or the corresponding D-amino of R acid substitution; . L at the 11 amino acid position of 1 or the corresponding D-amino of L acid substitution; . At the 12 amino acid positions of L°, 1 or W or 1 or W's reciprocal substitution with a corresponding D-amino acid; i F, Y, L or 1 at the 15 amino acid positions of W or F, Y, L or 1 substitution with a corresponding D-amino acid; o F, Y, L or 1 at 16 amino acid positions of W or F, Y, L or I* substitution with a corresponding D-amino acid; - D-amino corresponding to Q or Q9 at the 18 amino acid position of Y acid substitution; . The corresponding D-amino of R or R at the 20 amino acid position of K acid substitution; . At the 21 amino acid position of R, the corresponding D-amino of K or K acid substitution; i substitution of an amino acid with a corresponding D-amino acid.

LKKLYKRLVKILKRWWRYLKRPVR amino acid as defined herein A variant of the sequence is the D-amino corresponding to an L-amino acid itself. It may contain up to 24 substitutions with acid. Therefore, the variant sequence is completely It may consist of D-amino acids. For example, the variant sequence of an L-amino acid one or more of an L-amino acid itself with its corresponding D-amino acid said variant sequence with the substitution P139, as shown in Table 1. It contains at least 14 amino acids of an amino acid sequence of its peptides. More preferably, optionally the corresponding D-amino of an L-amino acid itself a polypeptide containing one or more substitutions with an acid, P1454, P148 or 02116-P-0001 It contains an amino acid sequence of the P159 peptide. In one direction, it is defined here In the figure, a variant sequence combines an amino acid itself with its corresponding D-amino acid. contains the substitution. The position of the D-amino acid in the amino acid sequence is irrelevant.

In another aspect, the variant sequence is the corresponding one of all L-amino acids themselves. It includes its substitution by D-amino acid. A variant as described here sequence of the amino acid sequence LKKLYKRLVKILKRWWRYLKRPVR or can be a reverse-evolutionary peptide of at least 14 consecutive amino acids of the sequence. Preferably, the word the variant sequence of said amino acid sequence, preferably P139-163 the complete amino acid sequence of one of its peptides, preferably P145, P148 or P159. is a reverse-inverting peptide of length. A back-evitic peptide, a reference amino It is a peptide composed of D-amino acids in the inverted sequence of the acid sequence. For example, A preferred variant sequence of the invention is the amino acid sequence of P145, P148 or P159. is the reverse-inverting peptide of the sequence, i.e., rvpkklyrylrkilkalrkylrkl, respectively, rvpkklqrwyrkllkfvrkwvrkl or rvprrlqryyrkllrfvrkylrkl strings or words has at least 14 amino acids from one of the sequences in question.

LKKLYKRLVKILKRWWRYLKRPVR amino acid as defined herein A variant of the sequence of an amino acid with an unnatural amino acid or after one or more of the amino acid substitutions shown above an unnatural expression of an amino acid present in said amino acid sequence may contain up to Sie substitution by amino acid. As used here, “natural- non-amino acids", irrespective of whether they appear in nature or not. refers to non-genetically encoded amino acids. Here may be present in a variant of an amino acid sequence as defined non-natural amino acids include: ß-amino acids; p-acyl-L- phenylalanine; N-acetyl lysine; O-4-allyl-L-tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine; 4-tert-butyl hydrogen 2-azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; 2,4-diaminobutyric acid; 6- aminocaproic acid; 2-aminoheptanoic acid; 2-aminois0butyric acid; 3- aminoisobutyric acid; 2-aminopimelic acid; p-aminophenylalanine; 2, 3-diaminobutyric 02116-P-0001 acid; 2, 3-diamino propionic acid; 2,2'-diaminopimelic acid; p-amino-L-phenylalanine; p-azido-L-phenylalanine; D-allylglycine; p-benzoyl-L-phenylalanine; 3-benzothienyl alanine p-bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosin; epsilon-amino hexanoic acid; N-ethylglycine; N-ethylasparagine; 2-IlorOphenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxylysine; allo- hydroxylysine; 3-(3-methyl-4-nitrobenzyl)-L-histidine methyl ester; isodesmosine; allo- isoleucine; isopropyl-L-phenylalanine; 3-methyl-phenylalanine; N-methylglycine; N- methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; methionine sulfoxide; 2-naphthylalanine; L-3-(2-naphthyl)alanine; isoserine; 3-phenylserine; norvaline; norleucine; phenylglycine; piperidinic acid; pyridylalanine; 1, 2, 3, 4-tetrahydro-isoquinoline-3- carboxylic acid; beta-Z-thienylalanine; y-carboxy-DL-glutamic acid; 4-fluoro-DL- glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy-tryptophan; -Ilor0-tryptofan; 3-Iloro-valine.

Preferably, a natural amino acid of said sequence is a corresponding natural- substituted by non-amino acid. As used here, a "corresponding “non-natural amino acid” is a natural- denotes non-amino acid. For example, a natural amino acid corresponding It is substituted by ß-amino acid. ß-amino acids are amino groups, natural amino acids as in acids (attached to [3 carbon instead of 1 carbon. For example, (i-alanine ß- substituted by alanine, etc. of a natural amino acid Other examples of its substitution by a natural-amino acid derivative are the following. Alanine eg beta-alanine, t-butylalanine, 2-naphthylalanine; L-3-(2-naphthyl)alanine, 2- replaced by aminoisobutyric acid. Arginine eg homoarginine, ornithine, N5- carbamoilomitin is substituted with 3-amino-pyronic acid. Asparagine eg N- substituted with ethylasparagine. Aspartic acid eg 4-tert-butyl hydrogen 2- substituted with azidosuccinate. Substitution of cysteine for example by cysteic acid, homocysteine is done. Glutamic acid such as y-carboxy-DL-glutamic acid; 4-fluoro-DL-glutamic acid is substituted. Glutamine eg D-citrulline is substituted by thio-L-citrulline. 02116-P-0001 Substitution by Glycine For example N-methylglycine, t-butylglycine, N-methylglycine, D-allylglycine Substitution of histidine for example by 3-(3-methyl-4-nitrobenzyl1)-L-histidine methyl ester is done. Substitution of isoleucine with e.g. isodesmosine, N-methylisoleucine, allo-isoleucine is done. For example, leucine is substituted by norleucine, desmosine, 5,5,5-trif10R0-leucine. Lysine eg 6-N-methyllysine, 2-aminoheptanoic acid, N-acetyl lysine, hydroxylysine, allo- substituted with hydroxylysine. Methionine is substituted, for example, by methionine sulfoxide.

Phenylalanine eg p-amino-L-phenylalanine, 3-benzthienyl alanine p- bromophenylalanine, p-acyl-L-phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4- substituted with fluorophenylalanine. Proline eg 3-hydroxyproline, 4-hydroxyproline, It is substituted by 1-acetyl-4-hydroxy-L-proline. Serine For example homoserine, isoserine, 3- substituted with phenylserine. Threonine e.g. D-thyroxine is substituted by allo-threonine is done. Tryptophan eg 5-hydroxy-tryptophan, 5-methoxy-tryptophan, 5-fluoro- substituted by tryptophan. Tyrosine eg O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 3- substituted with chloro-tyrosine. Valine eg norvaline, N-methylvaline, 3-fluoro0-valine is replaced with.

Therefore, here is the amino acid sequence LKKLYKRLVKILKRWWRYLKRPVR. or a polypeptide comprising a variant of said amino acid sequence The polypeptide in question is described as antimicrobial, antibacterial, antiviral, has anti-ingal, antiparasitic and/or anti-inflammatory activity, said variant sequence has at least 14 amino acids and is optionally has the following: . one or more of the following amino acid substitutions: 0 One or more selected from group L, V, F, A, 1, W, Y or Q of the amino acid with another amino acid selected from the group in question. or a corresponding amino acid of L, V, F, A, I, W, Y or Q substitution by non-natural amino acid; o where R° is K or K” with a corresponding non-natural amino acid substitution; 02116-P-0001 where K is R or K is a corresponding non-natural amino acid substitution; o one or more of an amino acid with a corresponding D-amino acid substitution. This type of variant sequence also includes a corresponding unnatural one or more amino acids selected from K and R substituted with amino acid A preferably, it may also contain substitution of at most one amino acid. Preferably, said variant sequence optionally using the following amino acid has one or more of its substitutions: K is R or an unnatural amino amino acid corresponding to R acid substitution; An unnatural amino amino acid corresponding to R° is K or K. acid substitution; L° is a corresponding natural- substitution with non-amino acid; A corresponding nature of the yin W or Q or W or Q° substitution by non-amino acid; A corresponding natural- substitution by non-amino acid; substitution; Winin F, Y, L or 1 or a corresponding number of F, Y, L or l its substitution with a non-natural amino acid; o one of an amino acid with a corresponding non-natural amino acid, or more substitutions.

More preferably, said variant sequence optionally with the following amino acid has one or more of its substitutions 02116-P-0001 At the 2 amino acid position of K, the corresponding non-native of R or R7 substitution by amino acid; At the 3 amino acid positions of K, the corresponding unnatural R or R° substitution by amino acid; At the 4 amino acid positions of L, V or the corresponding non-natural substitution by amino acid; At the 5 amino acid positions of Y, the corresponding natural- substitution by non-amino acid; At the 8 amino acid position of Ls, the V or the corresponding non-natural substitution by amino acid; At the 9 amino acid positions of V, the corresponding F or A or F or Asnin substitution with an unnatural amino acid; At the 10 amino acid position of K, R or the corresponding natural- substitution by non-amino acid; substitution by amino acid; 1 or W at the 12 amino acid positions of L or the reciprocal of 1 or W substitution by a corresponding non-natural amino acid; F, Y, L or 1 or F, Y, L or Isnin at the 15 amino acid positions of W substitution with a corresponding unnatural amino acid; F, Y, L or I or F, Y, L or Pnin at 16 amino acid positions of Ws substitution with a corresponding unnatural amino acid; At the 18 amino acid positions of Y, the corresponding natural- substitution by non-amino acid; At the 20 amino acid position of the canine, the corresponding natural- substitution by non-amino acid; At the 21 amino acid position of Ranin, the corresponding natural- substitution by non-amino acid; substitution of an amino acid with a corresponding D-amino acid. 02116-P-0001 A polypeptide, as defined herein from the amino acid sequence LKKLYKRLVKILKRWWRYLKRPVR or a part of this sequence may be a variant. As used herein, a "polypeptide" means more than one refers to amino acid containing peptides, polypeptides and peptidomimetics. The smallest polypeptide revealed to have antimicrobial activity is 14 amino acids. acid length. However, the amino acid sequence or variant thereof is larger. one or more additional amino acids at the N-terminus and/or C-terminus of a polypeptide may be part of an acid-extended polypeptide. amino acids of a polypeptide of the invention. acid sequence or variant thereof at the N-terminus and/or C-terminus, preferably an N-terminus and/or modified to include a C-terminal extension group. As an alternaive, said amino acid sequence or a variant thereof at the N- and/or C-terminus may be extended. Therefore, a polypeptide according to the invention contains at least 14 amino acids. and may contain up to 1000 amino acids. However, production costs are possible Smaller polypeptides are preferred for as low a record as possible. Preferably, more preferably 14-50 amino acids in length. For example, according to the invention a Contains amino acids. Preferably, the polypeptide is at least 16 amino acids, eg 16- It has amino acids. This type of polypeptide with 14-24 amino acids also contains an acet1-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2-O)ii-CO- and propionyl- such as an N-terminal modification selected from the group consisting of residue and/or amide-, consisting of NH-(CH2-CH2-O)1i-CO-amide- and one or two amino-hexanoyl groups an N-terminus and/or C-terminus such as a C-terminal modification selected from the group may have modifications. In one embodiment, a polypeptide of the invention is as defined, preferably P145, P148 or as shown in Table 1. from the amino acid sequence of P159 or at least 14 amino acids thereof formed, optionally having an N-terminal and/or C-terminal modification, preferably containing an N- and/or C-terminal extension group 02116-P-0001 from the amino acid sequence LKKLYKRLVKILKRWWRYLKRPVR or a part thereof consists of variant.

As used herein, “peptidomimetic,” non-peptidic structural elements antimicrobial of a polypeptide of the invention, containing antibacterial, antiviral, that mimics its antifungal, antiparasitic and/or anti-inflammatory properties. represents the compound. Thus, a polypeptide of the invention is non-peptidic. may contain structural elements. Such non-peptidic structural elements are a part of the invention. in the amino acid sequence of a polypeptide, one or more amino acids of said sequence may be present as a result of the substitution of the modification of the acid.

Alternatively, a polypeptide of the invention as defined herein LKKLYKRLVKILKRWWRYLKRPVR is outside of or outside the amino acid sequence in a variant, i.e. optional N- and/or C-terminal extension groups may contain non-structural elements. A non-peptidic in a peptidomimetic The structural element is typically a combination of one or more existing amino acids. modification. Preferred peptidoinimetics are, for example, described herein above. unnatural amino acids, conformer restrictions, polypeptide as Invent cyclization using isosteric replacement or other modifications It is obtained by structural modification of polypeptides. A polypeptide according to the invention amino acid sequence thus optionally one or more modifications includes. This type of polypeptide is produced by natural processes such as posttranslational processing or can be modified by chemical modification techniques. Modifications mention in the subject polypeptide, including the polypeptide backbone, amino acid side-chains It can be inserted in any position and at the N- or C-terminus. A single polypeptide contain more than one type of modification or many modifications to a single type can. Modifications include acetylation, amidation, acylation, phosphorylation, inethylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitin addition, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation and sulfation. In addition, according to the invention a polypeptide, with a label such as biotin, fluorescein, or flavin, with a lipid, or with a lipid 02116-P-0001 derivative, it can be equipped with a sugar group. A polypeptide according to the invention may also be a It can also be equipped with a targeting segment.

In a preferred embodiment, a polypeptide according to the invention is N-terminated and/or C- modified at the end. A polypeptide of the invention is thus preferably an N- and/or C- Includes tip extension group. N- and C-terminus that can be used in a polypeptide of the invention extension groups are well known in the art. The preference for an N-terminal modification Examples are an acetyl-, a hexanoyl-, a decanoyl-, a myristoyl-, an NH- It is (CH2-CH2-O)ii-CO- and a propionyl-residue. A C-end modification preferred examples are an amide-, an NH-(CH2-CH2-O)l-CO-amide- and one or two amino-hexanoyl group. However, other N- or C-terminal extension groups also will provide active compounds known to a person skilled in the art. A in practice, said polypeptide is an N-terminal acetyl, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2-O)ii-CO- or propionyl-residue and a C-terminal amide, It contains NH-(CH2-CH2-O)1i-CO-amide- and one or two amino-hexanoyl groups. A in practice, a polypeptide according to the invention is provided, where the N-terminus is acetylated and the C-terminus is amidated. Thus, here is a LKKLYKRLVKILKRWWRYLKRPVR amino acid sequence or an isolated or recombinant polypeptide containing a variant of the amino acid sequence The polypeptide in question is described as antimicrobial, antibacterial, antiviral, has antifungal, antiparasitic and/or anti-inflammatory activity, said variant sequence has at least 14 amino acids and optionally the following amino acids has one or more, preferably up to 10, of the acid substituents: 0 substitution of canine by R at amino acid position 2; o substitution of K at 3 amino acid positions with R; . substitution of L with V at 4 amino acid positions; - substitution of Y with W at the 5 amino acid positions; o substitution of L with V at 8 amino acid positions; - substitution of V at 9 amino acid positions by F or A; . substitution of K with R at the 10 amino acid position; 02116-P-0001 - substitution of I at the 11 amino acid position with L; . substitution of L with 1 or W at the 12 amino acid positions; . substitution of winin with F, Y, L or 1 at amino acid position 15; o substitution of W" at 16 amino acid positions with F, Y, L or 1; . ie substitution by Q at the 18 amino acid position; . substitution of K with R at the 20 amino acid position; i Substitution of R at the 21 amino acid position by K and/or . substitution of one or more amino acids with a corresponding D-amino acid wherein said amino acid sequence or said variant thereof, preferably an N-terminal acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2- O)i-CO- or propionyl-residue and a C-terminal amide-, NH-(CH2-CH2-O)i-i- CO-amide- and an N- and/or C- containing one or two amino-hexanoyl groups Includes tip extension group. Other N- or C-terminal extension groups will also provide active compounds. will be understood. Here, the numbering of amino acids follows The polypeptide is preferably P145, P148 as shown in Table 1. or an amino acid sequence of P159 or at least 14 amino acids thereof includes. Said polypeptide also preferably has 14-24 amino acids.

In a preferred embodiment, a polypeptide according to the invention includes a hydrophobic segment.

Addition of hydrophobic groups to cationic (poly)peptides, microbial endotoxin enhances their ability to neutralize and interact with microbial membranes and thus microbes. for example, it improves their ability to eliminate pathogens.

As hereinbefore described, a polypeptide according to the invention is known in the art. can be modified by chemical modification techniques. According to the invention modifications of polypeptides during or after the synthesis of the polypeptide 02116-P-0001 can be added. For example, using the polypeptide solid-phase synthesis technique When synthesized, the N-terminal acetylation of the amino acid sequence still attached to the resin can be carried out at the end of its reaction with acetic acid. another example C-terminal amidation, for example, in solid-phase peptide synthesis, is commercially available. A special type of resin such as Tentagel SAM (ex Rapp, Tübingen, Germany) carried out using These resins are formed during cleavage. Contains a chemical trap from which (poly)peptides are released. Modification of polypeptides These and other methods of known.

In a preferred embodiment, a polypeptide according to the invention penetrates a cell. Contains peptide. This type of cell penetrating peptide is an antimicrobial of the invention. Once bound to its peptide, the polypeptide acts across cell membranes. It is a peptide sequence that facilitates translocation. Any known in the art The cell penetrating peptide can be used in a polypeptide of the invention. To the cell Examples of penetrating peptides include, but are not limited to, polyarginine, TAT, HIV-Tat, R9-TAT, Pep-1, Pep-7, penetratin, transportan, Antp, Rev, FHV coat protein, buforin II, MAP, K-FGF, Ku70, SynB1, HN-1, TP10, pVEC, BGSC and BGTC are included.

A polypeptide of the invention is preferably a polypeptide that does not occur this way in nature.

That is, a polypeptide of the invention is preferably a non-naturally occurring polypeptide.

The expression "non-naturally occurring" as used herein means that the polypeptide not found in nature, preferably, the amino acid sequence of the polypeptide means not found.

In addition, one polypeptide of the invention comprising up to six polypeptides of the invention, A multimer containing the amino acid is provided. The multimer in question is the same up to six polypeptide monomers having the amino acid sequence or two or more six polypeptide monomers in which the excess polypeptide monomer has a different amino acid sequence 02116-P-0001 may contain up to polypeptide monomers. In a preferred embodiment, according to the invention bie multimer, up to six polypeptides according to the invention having the same amino acid sequence Salts of polypeptides according to the invention are also provided. These types of salts including, but not limited to, acid addition salts and base addition salts. Here as used, "pharmacologically acceptable salt" of a polypeptide the desired antimicrobial, antibacterial, antifungal, antiviral, that retain its antiparasitic and/or anti-inflammatory activity and or a salt suitable for administration to animals. Polypeptides Methods for the preparation of salts are known in the art and are generally of the polypeptide with a pharmacologically acceptable acid or base, for example the product one or more of the free acid or free base forms, the appropriate acid or base equivalent in a solvent or medium in which the salt is insoluble or subsequently dissolved react in a solvent such as water that is destroyed in vacuo or freeze-drying. a suitable ion exchange of cations of an existing salt or It involves mixing it by replacing it with another cation on the resin.

Examples of pharmacologically acceptable salts and bases, polypeptides formic acid, acetic acid, which forms ammonium salts with free amino groups, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and thiocyanic acid organic and inorganic acids such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine and other mono-, di- and trialkylamines and carboxylate salts of polypeptides such as arylamines with free carboxylic groups bases are included.

Polypeptides according to the invention can be prepared by various methods. For example, a polypeptide with widely used solid-phase synthesis methods, for example, good in the art by methods involving t-BOC or FMOC protection of known alpha-amino groups can be synthesized. Here, amino acids are a growing part of amino acids. 02116-P-0001 is added sequentially to the chain. Such methods are described, for example, by Merritield (1963), J. Am. in particular, a length of up to about 70 amino acids in large-scale production. Synthesis of polypeptides such as polypeptides or relatively short polypeptides It is suitable for

Alternatively, a polypeptide of the invention encodes the polypeptide well known in the art. recombinant techniques in which a nucleotide sequence is expressed in host cells can be prepared using The invention thus provides a polypeptide according to the invention. provides a method for its preparation, which includes: . a nucleic acid sequence encoding a polypeptide according to the invention. providing the nucleic acid molecule; . transforming a host cell with said nucleic acid molecule; . of said host cell to the expression of said polypeptide culturing under enabling conditions; i harvesting said polypeptide from said cells; . optionally at the N-terminus or C-terminus of said polypeptide, modified, for example, by adding an N- and/or C-terminal extension group to be made.

The invention also includes a nucleic acid encoding a polypeptide according to the invention, wherein a nucleic acid also referred to as a nucleic acid molecule according to the invention provides the molecule. As used herein, the invention is a nucleic acid molecule or nucleic acid sequence a chain of nucleotides, preferably DNA and/or RNA.

Also, a vector comprising a nucleic acid sequence molecule according to the invention is provided. As used herein, the term "vector" refers to a host cell's a plasmid, bacteriophage, or bacteriophage that can insert a heterologous nucleic acid sequence into 02116-P-0001 refers to a nucleic acid molecule such as an animal virus. According to the invention a vector of the invention by a heterologous nucleic acid sequence in a host cell. allows the expression or production of an encoded polypeptide. find it For example, a vector used according to its examples, not to be limited to these vaccinia (including Modified Vaksinia virus Ankara, MVA) virus, Newcastle Disease virus (NDV), adenovirus, or retrovirus derived from an animal virus. A vector according to the invention is preferably the chosen host. suitable for initiating transcription of a polypeptide of the invention in cells It contains an expression cassette containing a promoter. In eukaryotic host cells to examples of suitable promoters for the expression of polypeptides according to the invention, limited to the beta-actin promoter for mammalian hosts, immunoglobulin promoter, SS RNA promoter or cytomegalovirus (CMV), Virus such as Rous sarcoma Virus (RSV) and Simian Virus 40 (SV40) promoters derivative promoters.

Also, according to the invention, a nucleic acid molecule and/or a vector according to the invention A recombinant host cell containing it is provided. A host cell, according to the invention transformed by a nucleic acid molecule such as a vector, or It is a transformable cell. “Transforming” a recipient of a foreign nucleic acid Indicates insertion into the cell. Transforming a host cell by transient expression of a recombinant protein by that cell may result, i.e. the recombinant protein is expressed only for a defined period of time. is done. Alternatively, transformation of a recipient cell may result in stable expression. may result, that is, the nucleic acid is inserted into the genome of the cell and thus passed on to the next generation of cells. In addition, a recombinant protein inducible expression can be achieved. an inducible expression system, allowing expression of a nucleic acid sequence encoding a polypeptide of the invention. requires the presence or absence of a molecule that provides inducible Examples of the expression system include, but are not limited to, Tet-On and Tet-Off expression systems, for example an ecdysone inducible gene expression system, an arabinose-inducible gene expression system and an inducible 02116-P-0001 using a pMT/BiP vector (Invitrogen) containing the metallothionein promoter. Hormone inducible system such as the Drosophila inducible expression system are included. used in a method for preparing a polypeptide according to the invention. a host cell eg a Gram-positive prokaryote, a Gram-negative prokaryote or a eukaryote. Preferably, said host cell is a plant cell, a yeast. A eukaryotic cell, such as a cell, a mammalian cell, or an insect cell, most preferably an insect cell or a mammalian cell. Suitable host cells Examples are corn cells, rice cells, duckweed cells, (BY-2 or NT-1 cells) tobacco cells and plant cells such as potato cells are included. Examples of yeast cells are Saccharomyces and Pichi'a”d1. Bug Examples of cells from Spodoptera fmgiperda such as Tn5, SF-9 and SF-21 cells cells and Drosophila cells such as Drosophila Schneider 2 (SZ) cells.

Mammalian suitable for expressing a polypeptide according to the invention samples of African Green cells, but not limited to Monkey kidney (Vero) cells, baby hamster kidney cells (like BHK-21), Human retinal cells (eg PerC6 cells), such as (HEK293 cells) embryonic kidney cells, Madin Darby Canine kidney (MDCK) cells, Chicken embryo fibroblasts (CEF), Chicken embryo kidney cells (CEK) cells), blastodemia-derived embryonic stem cells (eg EB14), (3T3 cells) mouse embryonic fibroblasts, Chinese hamster ovary (CHO) cells and derivatives of these cell types.

A method according to the invention preferably further harvests polypeptides according to the invention. comprising a step of isolating, purifying and/or isolating. in hand Polypeptides according to the present invention are preferably used in human therapy, optionally additionally. from purification, isolation or processing steps, for example gel electrophoresis or used after purification using chromatography methods.

A polypeptide according to the invention has both therapeutic and non-therapeutic applications. exhibits a set of activities that can be used advantageously. In particular, according to the invention polypeptides, bacterial infections, fungal infections, viral infections 02116-P-0001 in inactivation of various microbial infections such as parasitic It is useful in neutralizing infections. Thus, according to the invention, a polypeptide or a pharmacologically acceptable salt thereof, and at least one containing pharmacologically acceptable carriers, diluents and/or excipients pharmaceutical compositions are provided. In addition, a nucleic acid according to the invention molecule or vector and at least one pharmacologically acceptable carrier, Pharmaceutical compositions containing diluents and/or excipients are also provided.

The invention also includes a polypeptide according to the invention for use as a medicine. it provides. In addition, a polypeptide according to the invention for use as a medicine A nucleic acid molecule comprising a nucleic acid sequence that encodes is provided.

Said drug may be a therapeutic or a prophylactic agent.

In one aspect, the specification includes a bacterial, fungal, viral and/or parasitic treatment of a subject suffering from or at risk of suffering from an infection for the subject subject of a polypeptide according to the invention, a pharmaceutical according to the invention. a therapeutically effective solution of the compound or a nucleic acid molecule according to the invention. discloses a method that involves the delivery of an amount of Also, with a microbe for the treatment of an infected subject or for the prophylaxis of a microbial infection Also disclosed is a method for preparing a medicament for a preferred In one aspect, said microbe is a bacterium, a fungus, a virus or a parasite.

In addition, a microbial, bacterial, fungal, viral and/or parasitic infection or from a microbial, bacterial, fungal, viral and/or parasitic infection use according to the invention in the prevention or treatment of a condition caused by A polypeptide and/or nucleic acid molecule is provided.

As used here, a "subject" is a human or an animal. to the subjects, Without being limited to these, humans, pigs, weasels, seals, mammals such as rabbits, cats, dogs, cows and horses, and chickens, ducks, including birds such as geese and turkeys. In a preferred embodiment of the invention, a the subject is a mammal. In a particularly preferred embodiment, the subject is a human. 02116-P-0001 The invention is also related to a microbe, such as a bacterium, a virus, a fungus or to prevent the growth of a parasite, the microbe or parasite in question comprising contacting a polypeptide or pharmaceutical composition according to the invention. The method also provides. Said contact may be in vivo or in vitro. realizable.

The polypeptides and pharmaceutical compositions according to the invention are used in various viral, bacterial and It is effective in the treatment of various microbial infections such as fungal infections. For example, polypeptides and pharmaceutical compositions, Gram-negative and Gram-positive It is effective in the treatment of bacteria. Whether in humans or animals which can cause infections that can be treated with polypeptides and their compositions. Samples of pathogenic bacteria, but not limited to Listeria, Eschericihia, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, Klebsiella, pseudomona, Legionella, diphtheria, Salmonella, bacillus, Vibrio cholera, tetanus, Clostridium, Bacillus, Yersinia and Leptospira bacteria are included.

Treatment in humans or animals with polypeptides and compositions of the invention Examples of pathogenic viruses that can cause infections but not limited to hepatitis A, B or C, herpes Virus (eg VZV, HSV-I, HAV-6, HSV-ll, CMV, EpsteinBarr-virus), adenovirus, influenza virus, Ilaviviruses, ecovirus, rhinovirus, coxsackie Virus, coronavirus, respiratory syncytial virus, HTLV virus, dengue virus, papillomavirus, polio virus, rabies Virus and human immunodeficiency virus (HIV virus; eg, types I and II).

Treatment in humans or animals with polypeptides and compositions of the invention Examples of pathogenic fungi that can cause infections, Without being limited to these, Candida (for example, albicans, krusei, glabrata, tropicalis), Aspergillus (for example, fumigatus, niger), Cryptococcus neoformans, Histoplasma capsulatum, Genus Mucorales, Blastomyces dermatitidi's, Paracocci'di'oides brasili'ensis and Coccidioi'des immitis are included. 02116-P-0001 Treatment in humans or animals with polypeptides and compositions of the invention Examples of pathogenic parasites that can cause infections but not limited to Entamoeba histolyti'ca, Plasmodium (e.g. falciparum, vivax), Entamoeba. Giardia, Balanti'di'um call', Acanthamoeba, Cryptosporidium sp., Pneumocysti's carini'i', Babesia microti, Trypanosoma (eg brucei, cmzi), Leishmania (eg donovani) and Toxoplasma gondi'i' are included.

In a preferred embodiment, the polypeptides and pharmaceutical compositions of the invention are methicillin-resistant Staphylococcus aureus (MRSA) and (non-resistant) S. aureuS, Staphylococcus epidermz'dz's, Gram-negative bacteria Pseudomonas aeruginosa and fungal species Candida albi'cans and Aspergillus nigersin cause It is effective in the treatment of infections.

Compositions containing polypeptides for prophylactic and/or therapeutic treatments can be given. In therapeutic applications, polypeptides or compositions are already a subject, preferably a human, suffering from the disease, symptoms and complications of the condition resulting from the infection is given in an amount sufficient to neutralize it. In prophylactic applications, polypeptides or compositions, contracting a microbial or parasitic infection to a subject at risk, for example a human or animal, to prevent infection or at least in an amount sufficient to prevent the development of an infection is given. The polypeptide is typically present in a pharmaceutical composition according to the invention or disease, especially associated with a microbial or parasitic infection in a therapeutic amount, an amount sufficient to ameliorate symptoms available. Typical doses of a given polypeptide according to the invention, or at least two combinations of dose, depending on the size of the polypeptide body weight It is between 0.01 and 10 mg per kilogram.

The polypeptides and pharmaceutical compositions of the invention are suitable for a wide variety of applications. suitable. For example, topical application can be used to treat eg skin infections, wound 02116-P-0001 in the treatment of infections and urinary tract infections or can be used to prevent Described here in detail As such, the polypeptides of the invention can inhibit biofilm formation and disperses the biofilms, the bacteria in and around the biofilm formation area, kills fungi or other microbes and pro-inflammatory microbial modulates immune responses by neutralizing endotoxins. bacterial microfilms can delay cutaneous wound healing and heal infected skin wounds, in curing skin infections or urinary tract infections or the topical antibacterial efficacy of conventional antibiotics in the treatment of can reduce. The invention is therefore associated with skin infection, wound infection. and/or use in the treatment or prevention of urinary tract infection for a polypeptide, pharmaceutical composition and/or nucleic acid molecule according to the invention it provides. In addition, a polypeptide according to the invention for use in wound healing, pharmaceutical composition and/or nucleic acid molecule are also provided. Also, leather treatment of infection, wound infection, urinary tract infection or a polypeptide according to the invention for inhibition and/or wound healing, use of the pharmaceutical composition and/or nucleic acid molecule is provided. The invention also protects from skin infection, wound infection and/or for the treatment of a subject suffering from a urinary tract infection, a polypeptide according to the invention, a pharmaceutical composition according to the invention or a therapeutically effective amount of a nucleic acid molecule according to the invention. It also provides a method that includes the given data.

Polypeptides of the invention can also be used for microbial, eg bacterial, viral, fungal, also advantageous as a preservative for materials susceptible to parasitic infection can be used in sequence. This type of material can be saturated with a polypeptide of the invention. or it can be covered or coiled. Described here in detail polypeptides of the invention are found in blood, plasma and serum, and plasma components It maintains antimicrobial activity in the presence of components such as Because The polypeptides and pharmaceutical compositions of the invention are particularly suitable for systemic administration. and treatment of infection associated with implants and medical devices, and/or 02116-P-0001 useful for prevention. As used herein, the term "medical device" means any type of verb that can be used on the human or animal body and to them. Without being limited to these, such instruments, medical tools, artificial joints, including hips and knees, and dental prostheses prostheses, chest implants, pacemakers, heart valves, stents, catheters, ear implanted tubes, such as splints, vias for medical devices, and wound or tissue dressings. available devices. Implants and medical devices often appear in Examples. successfully inactivated by the polypeptides of the present invention as with microbial infection, especially biofilm infections is related. In addition, implants and medical devices are often rapidly by plasma components from host fluids after implantation. hugs. Polypeptides of the invention are plazina as demonstrated in the Examples. Because it provides antimicrobial activity in the presence of its components, and/or microbial infection of medical devices a polypeptide according to the invention effectively treated and/or prevented by Therefore, find as a preservative for an implant and/or medical device. use is provided. In addition, microbial contamination of an implant and/or medical device infection, preferably bacterial infection, and/or A polypeptide of the invention is also provided for use in therapy.

A polypeptide of the invention advantageously has a controlled release and/or targeted joins the transfer carrier. As used herein, the term "controlled release" means denotes the release of the polypeptide of the invention in a time-dependent manner. A In practice, controlled release refers to slow release. used here As such, the term "targeted delivery" refers to a domain-directed expression of the polypeptide of the invention. means release. Use of a controlled release device Avoidance of frequent administration as by injection of the polypeptide of the invention has the advantage. The use of a targeted transmission means polypeptide in the body of a subject, an area of inflamation or an infection effectively transferred to an area of interest, such as has the advantage of being preserved. Preferably, a polypeptide of the invention 02116-P-0001 Microorganisms including bacteria, fungi, viruses and parasites It is targeted to an area infected by Controlled release and/or targeted transfer carriers are well known in the art. Controlled health and/or targeted non-limiting examples of delivery vehicles are nanoparticles, microparticles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes, serum albumin, antibodies, cyclodextrins and dextrans.

Controlled release, for example, of a polypeptide of the invention into such a carrier or It is provided by adding it to it. Carriers capture a polypeptide of the invention. form particles and such as aqueous, acidic or basic media or body fluids. polypeptide that slowly cleaves or dissolves in a suitable environment is made of releasing materials. Targeted transmission, for example, targeting on the surface obtained by providing a carrier with groups. This type containing targeting groups Carrier Examples antibody-domestic carriers, domain-specific ligand carriers and carriers with a positive or negative surface charge.

Particles of choice for controlled release and/or targeted delivery, nanoparticles optionally equipped with targeting groups. so in diameter particles from about 1 to 500 nm, preferably up to 200 nm, and are liposomes. Therefore, the invention includes a polypeptide of the invention and this type of controlled a controlled release carrier containing pharmaceutical compositions containing a release carrier it's on sale. In addition, a polypeptide of the invention and such targeted delivery a targeted delivery carrier comprising a pharmaceutical composition containing the carrier is also provided. Said carrier preferably consists of nanoparticles, from microparticles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes, serum from the group consisting of albumin, antibodies, cyclodextrins and dextrans is selected.

Preferred targeted delivery and/or controlled release carriers are biodegradable made of material. “biodegradable” as used herein, physiological denotes molecules that dissolve under certain conditions. These are hydrolytically soluble molecules and molecules requiring enzymatic dissolution. 02116-P-0001 Suitable biodegradable materials include, but are not limited to, PLA (poly lactic acid), PGA (poly glycolic acid), polycaprolactone (PCA), polyethylene oxide (PEO), polydioxanone (PDS), polycaprolactone (PCL), polypropylene fumarate, lactide, polymers derived from lactones such as glycolide and caprolactone, trimethylene carbonate and carbonates such as tetramethylene carbonate, dioxanones, ethylene glycol, polyester amide (PEA) ethylene oxide, esteramides, y-hydroxyvalerate, ß-hydroxypropionate, d-hydroxy acid, hydroxybuterates, hydroxy alkanoates, polyimide carbonates, polyurethanes. polyanhydrides and combinations thereof, hyaluronic acid, chitosan and cellulose biodegradable, such as polysaccharides and proteins such as gelatin and collagen polymers and natural biodegradable materials.

Also, preferably for implants and/or medical devices, use a polypeptide of the invention. A coating is also provided. In one embodiment, this type of coating It provides controlled release of polypeptide. This type of controlled release for medical devices The coating preferably contains a biodegradable material so that the invention Release of the polypeptide is achieved by dissolving the coating material. Because also a controlled release coating comprising a polypeptide of the invention. is provided. In addition, a polypeptide of the invention and a biodegradable material A medical device is provided containing this type of coating containing According to the invention a biodegradable coating, a biodegradable material as defined above includes. In particular, this type of biodegradable coating is from the group consisting of is chosen; PLA (poly lactic acid), PGA (poly glycolic acid), polycaprolactone (PCA), polyethylene oxide (PEO), polydioxanone (PDS), polycaprolactone (PCL), polypropylene polymers derived from lactones, such as famarate, lactide, glycolide and caprolactone, carbonates such as trimethylene carbonate and tetramethylene carbonate, dioxanones, ethylene glycol, polyester amide (PEA) ethylene oxide, esteramides, y-hydroxyvalerate, [3- hydroxypropionate, (i-hydroxy acid, hydroxybuterates, hydroxy alkanoates, polyimide carbonates, polyurethanes, polyanhydrides and combinations thereof, polysaccharides such as hyaluronic acid, chitosan and cellulose, and gelatin and collagen proteins. In addition, microbial infection of an implant and/or medical device, preferably for the prevention and/or treatment of bacterial infection, the word 02116-P-0001 a polypeptide of the subject implant and/or medical device containing a polypeptide of the invention. be provided with a coating and that the implant or medical device in question is attached to a subject. A method is provided that involves implanting it.

Polypeptides and pharmaceutical compositions also contain lipoteichoic acid, peptidoglycan and They neutralize pro-inflammatory microbial endotoxins such as lipopolysaccharides. and thereby the flux of neutrophils, macrophages/monocytes and lymphocytes and release of pro-inflammatory microbial compounds by the infected subject as anti-inflammatory agents because they prevent, reduce or inhibit is also useful. Also here is why the release of pro-inflammatory compounds According to the invention, a cell capable of releasing pro-inflammatory compounds A method of contacting the polypeptide is also disclosed. Aforementioned contact can be performed i'n in vivo and in vitro. Also here is an anti-inflammatory A polypeptide according to the invention is also disclosed as the agent.

Although polypeptides according to the invention are potent antimicrobial agents, such as antibiotics, antivirals and antifungals or other antimicrobial peptides with known antimicrobial agents such as conventional anti-infectives, antibodies and It can be combined with chemicals such as sensitizers, nanoparticles. this type a combination may result in an increased antimicrobial activity or can broaden the spectrum. Polypeptides of the invention, for example, bacterial with penicillins, cephalosporins, macrolides for the treatment of infections, with fluoroquinolones, sulfonamides, tetracyclines and/or aminoglycosides can be combined. For the treatment of viral infections, the polypeptides acyclovir, with antiviral nucleoside analogues such as ganciclovir, zidovudine (AZT), or didanosine or with neuramidase inhibitors such as oseltamivir, peramivir or zanamivir can be combined. Polypeptides of the invention for the treatment of fungal infections and compounds with polyene antifungals, imidazoles, triazoles, allylamines, May be combined with echinocandins, ciclopirox, flucytosine and/or griseofulvin.

The invention therefore includes a polypeptide according to the invention and preferably penicillins, A selected group of cephalosporins, carbapenems and mupirocin 02116-P-0001 containing an added antimicrobial agent such as an antibiotic or an antimicrobial peptide provides a pharmaceutical composition.

Pharmaceutical compositions according to the invention have at least one pharmacologically acceptable contains a carrier, diluent or excipient. Examples of suitable carriers are for example laparoscopic limpet hemocyanin (KLH), serum albumin (for example, BSA or RSA) and ovalbumin. In a preferred embodiment, the appropriate A carrier solution is, for example, saline. Tablets, capsules and the like Examples of excipients that can be added are: gum tragacanth, acacia, corn a binder such as starch or gelatin; an excipient such as microcrystalline cellulose; Corn starch, pregelatinized starch, alginic acid and the like disintegrating agent; a lubricant such as magnesium stearate; sucrose, lactose, or saccharin a sweetener such as; a flavoring agent, such as peppermint, oregano oil, or cherries.

When the dosage unit form is a capsule, in addition to the above types of materials, may contain a liquid carrier such as fixed oil. as tigers or otherwise various other means to modify the physical form of the dosage unit in various ways. materials may be available. For example, tablets can be mixed with lukewarm, sugar, or both. can be coated. A syrup or elixir active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, a dye, and cherry or orange It may contain a flavoring agent, such as a flavoring agent. A pharmaceutical according to the invention The composition is preferably suitable for human use.

The pharmaceutical compounds described herein can be administered in a variety of different ways. Examples include a pharmacologically acceptable polypeptide according to the invention. oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, given via intrathecal and intracranial methods. For oral administration, active ingredient in solid dosage forms such as capsules, tablets and powders or It can be given in liquid dosage forms such as elixirs, syrups and suspensions. 02116-P-0001 Sterile compositions for injection are, according to conventional practice, of the invention. polypeptide in water or sesame oil, coconut oil, peanut oil, rapeseed a naturally occurring vegetable oil such as oil, etc. or a product such as ethyl oleate or the like. by dissolving it in a vehicle for injection, such as a synthetic oil vehicle, or can be formulated by suspending Buffers, preservatives, antioxidants and so on may be included.

Compositions for topical administration are also according to conventional pharmaceutical practice. can be prepared. “Topical administration” as used herein means microbial or local treatment of conditions caused by parasitic infections refers to application to a body surface such as the skin or mucous membranes is doing. Examples of formulations suitable for topical administration, including but not limited to a cream, gel, ointment, lotion, foam, suspension, including spray, aerosol, powder aerosol. Topical medications can be epicutaneous, i.e. are applied directly to the skin. Topical medications can also be used, for example, in the respiratory tract. for administration to the epithelium of the mucous membranes, by inhalation or dermal can be applied to the surfaces of tissues other than, for example, applied to the conjunctiva eye drops or ear drops placed in the ear. topical administration said pharmaceutical composition formulated for preferably an emulsifier, a topical, such as a diluent, a humectant, a preservative, a pH adjuster, and/or water. comprising at least one pharmaceutical excipient suitable for administration.

A polypeptide according to the invention is also particularly suitable for diagnostic use.

Polypeptides are used to detect microbial infection, eg microbial toxins, such as blood, plasma, mucus, wounds, including LPS, LTA, and PG. of bacterial toxins present in the physiological sample, such as exudate and urine. can be used for detection. In addition, polypeptides are It can also be used to quantify microbial toxins in samples.

Because. a polypeptide nucleic acid according to the invention for use as a diagnostic agent. acid molecule is provided. In addition, blood, plasma, mucus, wound exudate and urine of a microbial toxin, preferably a 02116-P-0001 a polypeptide according to the invention for the detection of bacterial or fungal toxin. usage is also provided. As described above, according to the invention a the polypeptide is a biotin, a fluorescein tag, a near-infrared dye, or a It can be matched to a suitable segment, such as a radioactive isotp. This type is tagged detection of microbial infections such as polypeptides, bacterial infections It can be used in a method for migration because migration to an area of microbial infection they do. Using a detector suitable for the tag used attached to the polypeptide, It is possible to detect areas of infection. such as bacterial infections Methods for detecting microbial infections are therefore introduced by the invention. is provided. The method typically uses a microbial preparation of a labeled polypeptide. to a subject infected or suspected of being infected with the organism contains the given. The labeled polypeptide may interact with the infectious organism. accumulates at the site of infection. of microbial toxins in a physiological sample. The method is used to detect a labeled polypeptide in a microbial to a subject infected or suspected of being infected with the organism contains the given. Using various detectors sensitive to the tag attached to the polypeptide detect accumulation of the polypeptide at the site of infection or in a sample possible.

Another useful application of the polypeptides according to the invention is the use of food products. is protection. Therefore, furthermore, a food preservative of a polypeptide according to the invention It is also provided for use. In general, pathogenic or corrupt microorganism is the exposure of food to temperatures ranging from 60 to 100°C. It is destroyed by thermal processing. This process is on the food product, may have undesirable effects, such as undesirable organoleptic effects. find it The use of a polypeptide as a preservative in food products according to may result in increased lifespan and/or increased safety of the food product.

Pathogenic microorganisms in foods can cause infections or may cause poisoning and Campylobacter jejuni, Salmonella zfyphi, Salmonella paratyphi' and non-typhi Salmonella species, Staphylococcus aureus, 02116-P-0001 Escheri'chi'a call', Listeria monocyrogenes, Shigella and Clostridz my Botuli'num, Rotaviruses and viruses such as Norwalk virus, Taenia solium, Taeni'a sagi'nata and Contains parasites and lumps such as Trichi'nella spirali's. food spoilage, food change the appearance, consistency, flavor and/or odor of the products and Lactobacillus, Leuconostoc, Pseudomonas, Micrococcus, Bacteria such as Flavobaeterium, Serratia, Enterobacter and Streptococcus, Fungi and yeasts such as Aspergillus, Fusarium and Cladosporium can cause it. it could be.

The invention is more detailed in the following non-limiting examples. will be disclosed.

Brief description of figures w: 5 µM Cancidas or 0.8-6.4 µM in the presence of peptides shown Growth patterns of an A. nigefin cultured in PBS. Values, optical at 0 hours It is expressed as optical density at 600 nm according to the density. 16 Light of fungal growth in PBS after one hour of incubation micrograts. Representative light micrographs in triplicate.

Figure 2: 16 hours with PES, Cancidas, OP-145 or peptides shown Light growth of A. niger in 25% plasma after incubation micrographs. Representative light micrographs in triplicate.

SM: S. Biofilm formation at different concentrations (µM). Prevention by aureus JAR060131. Results, untreated ± standard of three independent experiments of biofilm mass relative to sample (0) expressed as the mean percentage of deviations. A, uncoated Biot'ilm formation in Kucuklai. B, biofilm in plasma-coated wells 02116-P-0001 Examples Materials and methods Synthesis of antimicrobial peptides Synthetic peptides are preloaded Tentagel resins for in situ activation. Using 20% piperidine in NMP for destruction of PyBop/NMM and Fmoc prepared with normal Fmoc-chemistry [Hiemstra HS et al. Proc Natl Acad Sci USA, carried out. After final Fmoc elimination, the peptides are C (triethylsilane) or containing additional sequestrants when present in the W (ethanethiol) peptide sequence. Cleaved with TFA/H2O 19/1 (v/V). Peptides by ether/pentane 1/l (v/v) precipitation and isolated by isolation of the product by centrifugation. With air at about 40°C After drying, the peptides were dissolved in acetic acid/water 1/10 (v/v) and was lyophilized. Peptides Purity using UPLC-MS (Acquity, Waters) Maldi-Tof mass spectrometry showing expected molecular masses in terms of (Microflex, Bruker) checked for integrity.

Abbreviations: Fmoc: 9H-f10renylmethyloxycarbonyl NMM: N-methylmorpholine PyBOP: Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate TFA: trichloroacetic acid bacterial strains clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) LUH14616, Dr. S. Croes, Maastricht University Medical Centre, Maastricht, 02116-P-0001 S. aureus JAR, Campoccia et al. (Int .I Artif Organs. 2008 Sep;3l(9):84l-7) is described. is being done.

It is described in D60°.

Bacteria were stored at -80°C until use. intermediate stage bacteria inoculum of bacterial colonies isolated from blood agar plates for 2.5 hours. On Tryptic Soybean Water (TSB) medium (Becton Dickinson, Le Pont de Clax, France) It was prepared by incubating and then adjusted to the needed concentration. Determination of antibacterial activity Peptides S. aureus JAR in PBS LUH14616, Staphylococcus epidermis's RP62a and Pseudomonas aeruginosa concentration of pooled human plasmin (Sanquin, Amsterdam, The Netherlands) incubated with or without addition. Antimicrobial activity 99.9% fatal concentration (LC99.9), i.e. 2 hours under shaking conditions at 37°C the lowest peptide that kills 99.9% of bacteria after incubation expressed as concentration.

Determination of antimicrobial activity of peptides against dormant stage bacteria The peptides were prepared for 18-20 hours under the conditions described above. 02116-P-0001 was incubated with.

Determination of antifungal activity Peptides 1X105 of a mid-logarithmic culture of Candida albicans Y-01 pooled human plasma at a final concentration of 50% by cells/ml incubated with or without addition. Antimicrobial activity 99.9% fatal concentration (LC99.9), i.e. 2 hours under shaking conditions at 37°C the lowest peptide at which 99.9% of cells were killed after incubation expressed as a concentration.

Effect of peptides on fungal growth assay using Aspergillus niger. was done. Peptides 7.5 of A. niger PagsA-lux in PBS with 104 spores/mFS, 25% with or without the addition of pooled human plasma at a concentration incubated. Antifungal caspofungin (Cancidas) to spores as a positive control. was treated with. Absorption was measured in the course and at 16nc1 hour, fungal Growth was visualized using light microscopy.

Determination of anti-bivofilm activity In the figure, biofilm-adjusted BM2 in 96-well polypropylene plates After incubation, planktonic bacteria were destroyed by four washes with PBS and biofilms were stained with crystal purple. After solubilization with ethanol, at 590 nm optical density was determined as a measure of biofilm mass. Anti-biophilic activity is 50% inhibitory concentration (ICSO), i.e. 2% 50% of the biofilm mass expressed as the lowest peptide concentration resulting in reduction is being done. 02116-P-0001 For the determination of anti-biofilm activity of peptides in the presence of plasma, 96- polypropylene well plates overnight incubation with 20% plasma at 4°C was made and coated with plasma. The wells were washed once with sterile water and infused with S. aureus and peptides as described.

Immunomodulatory activity: LPS and LTA neutralization Peptides or 2 mg/ml LTA (S. aureus, endotoxin free) or 1x]09 CPU/ml with UV-killed S. aureus JAR060131 It was pre-incubated at 37°C for minutes. Diluted whole human blood 20 hours peptide-LPS/LTA/S at 37°C for a period of time. aureus was stimulated with mixtures. in the filtrate neutralizing activity 50% or 90% inhibitory concentration (IC50 and IC90), expressed as the lowest peptide concentration resulting in reduction is being done.

Results Identification of 25 peptides derived from OP-145 Antimicrobial and less sensitive to plasma components than OP-145 The new peptides, which were peptides, were identified by computer prediction.

The OP-145 is predicted to adopt an amphipathic spiral structure. This type of In structure, the peptide has charged groups on one side of the helix and hydrophobic on the other. folds in an a-helix containing groups. Have amino acid substitutions From previous studies applying peptides, the hydrophobic side of the helix on the loaded side of the helix or addition of charged groups resulting in The addition of hydrophobic groups resulting in reduced antibacterial activity We know that this results in compounds with 02116-P-0001 Thus, the newly designed peptides are folded in an amphipathic helix. should be foreseen. Based on the sequence of OP-145, for amino acid substitution We designed the motif below. By minimizing the structural similarity to the OP-l45 and anti- minimizing binding to plasma components affecting microbial activity. Peptides significantly separate from OP-145°, maximizing the chance of being Computer prediction, peptides with an amphipathic helix of the following motif The following 25 peptides were chosen based on the motif (J = acetyl, B = amide). 02116-P-0001 acetyl, 8 Table 1. Sequence of P139-163 peptides. J W K R V F R I W K R I F R Y L K R P V R B W K R L V R I I K R I Y R Q L K R P V R B P139 J L K K L P14O J L R R L P141 J L R R L Y K R V F R L L K R W W R Y L K R P V R B P142 J L R R L P143 J L R R L Y K R V V K L W K R L F R Q L R R P V R B P144 J L K K L Y K R V A K I W K R W I R Y L K K P V R B P146 J L K K L Y K R L F K I L K R I L R Y L R K P V R B P147 J L K K L W K R L A R L L K R F I R Q L R R P V R B P148 J L K R V W K R V F K L L K R Y W R Q L K K P V R B P149 J L K K V Y K R L A R L K R Y I R Y L R R P V R B P15O J L K K V W K R V A R L I K R W F R Y L R R P V R B P151 J L K K L Y K R L F K L W K R L Y R Y L K K P V R B P152 J L R R V Y K R L A R L 1 K R Y L R Q L K K P V R B P153 J L R K L W K R V V K 1 W K R Y L R Q L R R P V R B P154 J L R K L W K R L A K I I K R L Y R Y L R R P V R B P155 J L K K V Y K R V A R L I K R L F R Y L K R P V R B P156 J L R R L P157 J L K K V W K R V F R I L K R F L R Y L K R P V R B P158 J L R R V Y K R L F R L W K R I I R Q L R R P V R B P159 J L K R L Y K R V F R L L K R Y Y R Q L R R P V R B P160 J L K K L W K R L A R L W K R 1 1 R Q L K K P V R B P161 J L R R V W K R V A R I I K R L Y R Y L K R P V R B P162 J L K R L W K R L F K I L K R Y Y R Y L R R P V R B P163 J L R R L W K R V F K I I K R L F R Q L K K P V R B These peptides have been shown to have antibacterial activities in the absence and presence of plasma. tested (Table 2). were selected as value-enhanced peptides.

Table 2. Antimicrobial activities of peptides derived from OP-145. lAn'thymicrobial activity lC99.9 (µM), i.e. within 2 hours (approximately 1x106 CPU/ml (which is S. aureus JAR) is the most abundant, killing 99.9% of bacterial inoculums. expressed as the low peptide concentration. 02116-P-0001 Peptide Antimicrobial activity IC99.9 (pM) PES 50% plasma 02116-P-0001 Peptide Antimicrobial activity1 IC99.9 (µM) PBS 50% plasma Antimicrobial activity of P145, P148 and P159* against different bacteria In PBS, P145, P148, and P159 were mid-logarithmic to all bacterial species in culture. had a similar antimicrobial activity against OP-145 (Table 3). 50% (for S. epidermis'di's RP62a (43-fold) and P. aeruginosa PAO1 (>11-21-fold) higher bactericidal showed activity. antimicrobial activity in logarithmic cultures. The results are LC99.9, so, lowest peptide (in µM) resulting in :999% killing of bacteria expressed as a concentration. Results, at least two independent attempts are the median values. 02116-P-0001 LC99.9 (pM) aureusJAR060131 P1451.6 19.2 P1481.6 19.2 P159 2.4 12.8 1.6 204.8 1.6 9.6 1.6 8.0 2,4 9.6 epidermis'disRPöZa 0.8 204.8 0.8 3.2 0.8 3.2 1.6 3.2 aeruginosaPAOl 3.2 >204.8 1.6 25.6 1.6 12.8 1.6 12.8 aureus and P159 compared to logarithmic bacteria It showed similar antimicrobial activity against JAR060131 (Table 4). Thus, antimicrobial activity against the suspension. Results in three independent experiments are the average values.

LC99.9 (µM) >204.8 02116-P-0001 Antimicrobival activity of length variants of pl48 Deletion of 4 amino acids at the C- and N-terminus of P148 versus S. aureus JAR06013l had no effect on antimicrobial activity (Table 5). C- and N- deletion of 5 amino acids at their ends reduces antimicrobial activity compared to P148 however, activity still increased 5-fold compared to OP-145.

Table 5. P148 length variants in PBS and 50% plasma antimicrobial activity. Results are LC99.9, ie S. aureusaun 2%99.9% as the lowest peptide concentration (in µM) resulting in the killing of is expressed. The results are the mean values of two independent experiments. J = acetyl, Peptide Sequence JLKRVWKRVFKLLKRYWRQLKK JRVWKRVFKLLKRYWRQLKKPV JLKRVWKRVFKLLKRYWRQLKK JRVWKRVFKLLKRYWRQLKKPB JWKRVFKLLKRYWRQLKKPVRB JLKRVWKRVFKLLKRYWRQLKB JVWKRVFKLLKRYWRQLKKB JWKRVFKLLKRYWRQLKB JKRVFKLLKRYWRQLB LC99.9 (µM) 02116-P-0001 Antimicrobial effects of P145, P148 and P159 with multiple alanine substitutions activity substitution had no effect on antimicrobial activity against S. aureus JAR060131. has no effect (Tables 6-9). 02116-P-0001 Table 6. P'i45's. With the Cam of a field with different Positions in PES AND 50% plasma antimicrobial adj. Two independent ::e 13"`m results bee. .J = acetyl, 3 = amide 24'-- 1755-04 1255 05 1255 07 175310 2235-11 1255 20 115&)1 1255-22 ”5521 1155 2:! 1255-25 375925 ”55.27 1255-28 iiKRiviRiAkiitü .kRRIV'iRLAKL 'i-'iLV .LKALT'RELAKL›I.-1LV iKRiAxaiAKi xâiv Lxnivxaiisiiquiv LKRIVmaLvki=n=Lf iKniviuiAin iaiv LKRlVLJLAKLAEFLV lKRlYKHlAKî «niv LtniVKHLAxizn4Av itKRiYKHLLAKL ua ilKR: to the KRLANL JLKRL KRLAKL MH KRLAKLIKH 12'.- Table 7 of P148. PBS v is an area of an area at different positions in 50% plasma antimicrobial activity by substitution Results of two independent experiments. = acetylv 8 = amide. 175510 1255 33 1755-94 1255!. 1255 &3 2155-4' 1155-43 2255 4'3 .255 50 2255 S? .IKRK'Ã-RGVV 17Ã^\'\$'Â.Q ; yi; i '.'Nv I H' ' 4, R 'i".\'›: il L' › 1 Rw.' v: kit'xîß`iii H '1 V.' 51 9 V' '1-' 51 `1 `1. 517%: P 1W›› 7' V'I'î' F 1 7.- u 1!) I) 1:50:50 :) m .sospvlazmn _'58_ 5? plasma IKPu'R* 15 'itv l& '-3` nun: ;ie sýirz k 0; llii npvki *in %1: 4 , @gr , :Su april iis 512M izs XPh'R'r 15 ; si: :c 12: (Pvkh ii, in ;6 125 lKKPvP.? _La . :56 .16 AKKFVRL Lrs. 08 zan ounce 201.: 01 is __ 929.? I-; m 12'» PiRr. p .'55 ci:: M: iiiin lp gs:: :34 PVREi _ ;L _ :SÇ _.6_ LIG P'vRi':e 255 ar; Shiite PVP.:` :li 25-. :-3 bi) FRP; :o *il 173 7 15: PR'II:: 11: J'iS LC› 13':- PK'RE lli :'.iu 115 9351- ]m Sl) I?" LC999 'IM' 02116-P-0001 Table 8. P159. A mixture in PBS and 50% plasma at different positions. antimicrobial activity by alanine substitution. Try two independent results.

J = acetyl, 8 = amide. Lcsas (um) .

Peptide Ca"$ma* Sequence ms 50% .plasma Pas 50% plasma Table 9. P148”in. Antimicrobial activity with multiple alanine substitutions in PBS and 50% plasma.

The results are the mean values of two independent experiments. J = acetyl. B = amide.

LC99.9(11M) Peptide PBS 50°..

SERI plasma 02116-P-0001 Antimicrobial effects with positive amino acid substitutions of Pl48 variants activity Variants of Pl48 in which lysine or arginine is replaced by a positively charged amino acid. antimicrobial against S. aureus both in PBS and in the presence of 50% plasma retains activity (Table 10).

Table 10. P148 with positively charged amino acid substitutions in PBS and 50% plasma antimicrobial activity. J = acetyl, O = ornithine; X = diaminobutyric acid (DABA): U = diaminopropionic acid (DAPA), B = amide.

LC99.9 lEMl Peptide SEQUENCE PES 50% plasma P148 LKR1/'E'JKRVFKLLKRY QLKKF'IR' 0.8 12.8 P365 '.<`3R`~/WKR`JFI-Ç_'_KRE Q_l'-'.KP'/P 0.8 12.8 PBGG '_ Ki`.r'\fi*.'KR"J Fl-I. _ _ KR& Q _l-(KP'JR 0.8 12.8 P367 LKRVih"R"/FKLLKRY' is 'QLKKP'. 0.8 12.8 P369 ._ K R“iih'K RVFÖ L _ KRY "ELK K PVR 0.8 12.8 P370 7 KRVW KRVFKl _iÂFRY QLK KPIIR 0.8 25.6 P371 _KRVWKRVFKL_KQ`~ ;`g_l\'KP'/R 0.8 25.6 P373 :KRV WKRVFI-ÄL L KR" `j ,CHP R 0_8 25.6 P374 ;KR'IWKRVFKL_KR 'QLKîêP` 0.8 25.6 P375 :KRVWKRVFKLIKR QLKKP'IÜ. 0.8 12.8 P377 L.I«î`R'JirlKR`J}-K~_KR ';gnl\IKP'/R. 0.8 12.8 P381 LKR'Iii'JKR'IFF L L KRY -g KKF'IR 0.8 25.6 P382 :KRVWBRVFKLLXRY is QLKKP. 0_8 25.6 P383 LKRVWKRVFKL _KEY 'QLKKP'JR- 0.8 25.6 P38S ;KRVWKRVFKL_KRY QLNKP'IR': 0.8 12.8 P386 LKRVWKR'JFKL_KR` "ELKXP'JR' 0.8 25.6 P387 LKRVWKRVFKLLKR QLKKP'IX 1.6 25.6 P389 :ÜPVWKR'IFKLLKR QIKKP'IR': 0.8 12.8 PSSD I.K:`Ji'.'KR'JE-EL_KR QLI'IIKP'JR` 1.6 12.8 P391 LKR'IL'CIRVFKLLKR QLKKP'IRÄ. 0.8 25.6 P392 LKR"-/"wlx"7FK'__E\R ;ILKKP'JRI 0.8 12.8 P393 LKRV'»'JKR"/Fi"`L KR QLKKP'IRI 0.8 25.6 P394 ,KRVV'BRVFKLLUR iQLE-(KP/R 0.8 25.6 P397 LKRVWKRVFKLLKRY' QLUKP'IR: (18 25.6 P398 ;KR'IV-'KRVFKL;KRYWRQLKIGPVR 1.6 25.6 P399 ;KP'f'i'KRVFKL_KRYWRQLKKPVÜ 3.2 51.2 02116-P-0001 Antifungal activity of P145, P148 and P159 OP-145 at 51.2 µM in PBS 'no antifungal activity against C. albi'cans Y-Ol' (for P148) 99% of C. albicans at concentrations ranging from it happened. At this concentration, no antifungal activity was observed for OP-145. activity. The results are the mean values of two independent experiments.

LC99 (iiM) PES 50% plasma OP-145 inhibited A. niger growth by >99.9% at concentrations of 3.2 µM (Fig. one). P145 showed similar antifungal activity as OP-145, whereas P148 showed similar antifungal activity. 4-fold higher antifungal activity while inhibiting growth already at 0.8 µM° he had. P159 has a 2-fold lower antifungal activity compared to OP-145°. he had. As the plasma affects the optical density values, the plasma peptides Its antifungal activity can be determined based on light micrograms only. was done. At 25% plasma presence, fungal growth was inhibited by 204.8 µM of OP-145 02116-P-0001 Anti-bivofilm activity of P145, P148 and P159 biofilm inhibition was approximately 75%. Notably, biofilm-adjusted In BM2 media, these peptides did not show antimicrobial activity up to 51.2 µM.

In plasma-coated wells, 3.2 µM of OP-145 and P159 inhibited biofilm formation. and a two-fold higher concentration was required for P148 (Figure 3B). Plasma changed between

Immunomodulatory activity: LPS and LTA with P145, P148 and P159 neutralization prevented. Ninety percent of LPS-induced inhibition of IL-12p4031n, from P148 to The ability to neutralize LTA is mediated by blood cells for LTA-induced IL-8 production. determined by measuring prevention. At a final concentration of 0.781 µM, P159 had a 4-fold increased LTA-neutralization capability. Peptides are also described in S. aureus JAR06013liin was pre-incubated with UV-killed bacteria. OP-145” Incubation with 0.195 µM of IL-8 production induced by S. aureus JAR has neutralizing activity, while >50% of S. aureus-triggered IL-8 production An 8-fold higher concentration of P145 and P148 required (Table 12). 02116-P-0001 activity. For LPS neutralization, the experiment was performed using blood from two donors. carried out. For LTA and S. aureus neutralization, the test was obtained from a donor. performed using blood.

Peptide LPS neutralization (IC90 in nM) P145 0.75 P148 0.25 P159 0.25 LTA neutralization (IC90 in nM) 0.781 0.195 0.195 0.195 aureus neutralization (nM as IC90) 0.195 1,563 1,563 0.195 02116-P-0001 RzLâGSWm 9.1 0" 0944:& 0 8 pw! 1.6;1M . cmcicias 3 4 ;5 g graduated Duration (hours) 4 of S 10121(16 Duration (hours) 02116-P-0001 0.10* 0.35" 0.8"M 4 6 3 *O '%2 '%4' 0» ;JM Duration (hours) 0.8 m 1.::: pm 3 214%', Duration (hours) Figure 1, continued 02116-P-0001 /14 ;74 02116-P-0001 02116-P-0001 9193515 1 02116-P-0001 P145 9148 Or-i-ucöoi ::ic-'05:60:' civ-edco'oi P148 P159

Claims (5)

CLAIMS An isolated or recombinant polypeptide containing a LKKLYKRLVKILKRWWRYLKRPVR amino acid sequence or comprising a variant of said amino acid sequence, said polypeptide having antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic activity and against at least one microbial species in the presence of 50% plasma , when detected under the same conditions, OP-145 (acetyl- has a high in vitro antimicrobial, antibacterial, antiviral, antifungal and/or antiparasitic activity, said variant sequence has at least 14 amino acids and optionally has: - the following amino 1 to 8 traces of acid substitutions - substitution of one or more amino acids selected from the group L, V, F, A, I, W, Y or Q with another amino acid selected from said group; one or more substitutions with an amino acid; - one or more substitutions of an amino acid with a corresponding non-natural amino acid, said corresponding g the eliminated unnatural amino acid is a derivative of the reference natural amino acid; and/or - a reverse-inverted sequence of at least 14 consecutive amino acids from said amino acid sequence, characterized in that said variant sequence has at least one KRLVKILKRWWRYL amino acid sequence optionally having one or more of the amino acid substitutions as defined above. 2. A polypeptide according to claim 1, characterized in that said one or more substitutions of an amino acid with a corresponding non-natural amino acid is selected from the group consisting of: o substitution of one amino acid by the corresponding 6 amino acids; o substitution of R0 by homoarginine, ornithine, N5-carbamylornithine or 3-amino-propyronic acid; o substitution of I by isodesmosine, N-methylisoleucine or allo-isoleucine; o substitution of L with norleucine, desmosine or 5,5,5-triH0r0-leucine; o substitution of K by 6-N-methyllysine, 2-aminoheptanoic acid, N-acetyl lysine, hydroxylysine or allo-hydroxylysine; o substitution of P by 3-hydroxyproline, 4-hydroxyproline or 1-acetyl-4-hydroxy-L-proline; o substitution of W by 5-hydroxy-tryptophan, S-methoxy-tryptophan or S-fluorotryptophan; o substitution of Y by O-methyl-L-tyrosine, O-4-allyl-L-tyrosine or 3-k1010-tyrosine; and o substitution of V7 by norvaline, N-methylvaline or 3-fluoro0-valine. 3. Polypeptide according to claim 1 or claim 1, characterized in that said polypeptide is optionally modified to N-terminus and/or C-terminus, preferably an N-terminus acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-( It contains a CH2-CH2-O)ii-CO- or propionyl- residue and/or contains a C-terminal amide-, NH-(CH2-CH2-O)ii-CO-amide- or one or two amino-hexanoyl groups. 4. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1-3. 5. A vector comprising a nucleic acid molecule according to claim 4. A recombinant host cell comprising the nucleic acid molecule according to claim 4 and/or the vector according to claim Si. Polypeptide according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, nucleic acid molecule according to claim 4 and/or vector according to claim Si, and at least one pharmacologically acceptable carrier, diluent and/or excipient It is a pharmaceutical composition containing The pharmaceutical composition according to claim 73, further comprising an additional antimicrobial agent preferably selected from the group consisting of penicillins, cephalosporins, mupirocins and carbapenems. The pharmaceutical composition according to claim 7 or 8, characterized in that it contains a controlled release and/or targeted delivery carrier containing said polypeptide, so that said carrier is preferably composed of nanoparticles, microparticles, nanocapsules, microcapsules, liposomes, microspheres, hydrogels, polymers, lipid complexes. is selected from the group consisting of serum albumin, antibodies, cyclodextrins and dextran. A coating for a medical device comprising a polypeptide according to any one of claims 1-3. For use as a therapeutic, prophylactic or diagnostic agent which is a polypeptide according to any one of claims 1-3 and/or a nucleic acid molecule according to claim 4. Polypeptide and/or nucleic acid molecule for use according to claim 11 in the treatment of a microbial, bacterial, fungal, viral and/or parasitic infection and/or in the treatment of a condition resulting from a bacterial, fungal, viral and/or parasitic infection. 13. A polypeptide and/or nucleic acid molecule according to claim 11 or 12 for use in the treatment and/or prevention of biofilm-associated infection. 14. A method for preparing a polypeptide according to any one of claims 1-3, characterized in that: - providing a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide according to any one of claims 1-33; - transforming a host cell with said nucleic acid molecule; - culturing said host cell under conditions allowing its expression with said polypeptide; - harvesting said polypeptide from said cells; optionally modifying said polypeptide as N-terminus or C-terminus.